Abstracts of papers presented at the Sixth International Verticillium Symposium Abstracts of papers...

MEETINGS

ABSTRACTS OF PAPERS PRESENTED AT THE

S I X T H I N T E R N A T I O N A L VERTICILLIUM S Y M P O S I U M

June 19-23, 1994 Hod Hotel, Dead Sea, Israel

A: DETECTION, IDENTIFICATION, DJFFERENTIATION AND CHARACTERIZATION OF VERTICILLIUM BY MODERN METHODS

VerticiUium-Species-Specific Molecular Probes and Their Application for Biological Assay and Phylogenetic Studies

L. Kening, D.I. Rouse and T.L. German Dept. of Plant Pathology, University of Wisconsin, Madison, W153706, USA

[Telefax: +1-608-263-2626]

Detection and quantitation of plant pathogens is essential for improved understanding of their role in the epidemiology of disease. Molecular probes may provide a tool for unambiguous identification and enumeration of Verticillium species. Polymerase chain reaction primers were identified that amplify a 700 base pair (bp) region of the mitochondrial rRNA gene of Verticillium. Based on tests with nine species of Verticillium and 60 isolates of V. dahliae, these primers were shown to bind to sites that were conserved across the genus. Although the priming sites were conserved, the region amplified was variable between species. By cloning and sequencing the amplified region, probes specific for V. dahliae, V. albo-atrum and I,'. tricorpus were identified. In a second approach, random amplified polymorphic DNA (RAPD) was used to obtain a 600 bp fragment of DNA that was shown to be unique to V. dahliae. Further examination of RAPD data from 32 isolates of V. dahliae indicated that vegetative compatibility group (VCG) 1 was a separate

Phytoparasitica 23:1~ 1995 39

and unique group, while the other VCGs were similar to one another. It was possible to identify subgroups within VCGs 2, 3 and 4, but they did not necessarily match the vegetative compatibility groupings. (L)*

Genome Analysis in Isolates of Verticillium, V. albo.atrum, 11. dahliae and V. lecanii

D.M. Roberts, 1 B.W. Bainbridge, t H.C. Evans 2 and J.B. Heale 1 1Microbial Physiology Research Group, King's College, Kensington,

London "W8 7AH [Telefax: +44-71-333-4500]; and 2International Institute for Biological Control, Ascot SL5 7TA, UK

Genomic DNA from 44 isolates from the fungal genus Verticillium was amplified using single primers and the polymerase chain reaction-based technique known as Random Amplification of Polymorphic DNA (RAPD) to generate reproducible fingerprints. Of these isolates, 19 were of the entomopathogenic species V. lecanii and the remaining were of the plant pathogenic species V. albo-atrum (15) and V. dahliae (10). After separation by gel electrophoresis, the amplification patterns were analyzed using UPGMA (unweighted paired-group matching analysis) cluster analysis to determine if the species could be separated at the molecular level based on DNA polymorphisms detected by the RAPD technique. The cluster analysis revealed a high degree of similarity between the V. albo-atrum and the V. dahliae isolates. It also showed the species V. lecanii to be a very heterogeneous group which also displayed a high degree of dissimilarity to both V. albo-atrum and V. dahliae. This low degree of similarity between the V. lecanii isolates and the other Verticillium species analyzed indicates a wide taxonomic difference between these species. (L)

RAPD Analysis of VerticiUium dahliae in Five Different Pathogenicity Groups in Japan

M. Koike, 1 M. Watanabe 1 and H. Nagao 2 1Dept. of Agro-environmental Science, Obihiro Unhpersity, Obihiro, Hokkaido,080

[Telefax: +81-155-474020]; and 2Laboratory of Plant Pathology, Faculty of Horticulture, Chiba University, Matsudo, Chiba 271, Japan

Verticillium dahliae is generally known as a pathogen with a wide host range. In Japan, host specificity has been recognized in tomato, sweet pepper and cruciferous vegetables such as Chinese cabbage and Japanese radish. Horiuchi et al. (1990) and Hagiwara (1990) established five pathogenicity groups in V. dahliae isolates in Japan: those pathogenic to eggplant and turnip (group A, eggplant strain); to eggplant, tomato and turnip (group B, tomato strain); to eggplant, sweet pepper and turnip (group C, sweet pepper strain); to turnip alone (group D, crucifer strain); and a less pathogenic strain (group E). However, it is very laborious and time-consuming to identify these pathogenicity groups, and simple methods are needed for identification. RAPD (random amplified polymorphic DNA) analysis is the most recent addition to the arsenal of biotechnology- based methodologies that can be applied to basic studies of genetic relationships between taxa. The aim of our research was to identify five pathogenicity groups of V. dahliae using RAPD analysis. DNA polymorphisms between five groups were detected in 12 of the 20 primers used. The patterns

*L = lecture sessions; P = poster (market place) sessions.

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were ABC/DE, AC/B/DE, A/BC/DE, A/B/C/DE and AC/B/D/E. Five groups could be identified by RAPD analysis. (L)

Use of Random Amplified Polymorphic DNA (RAPD) to Characterize Cotton-Defoliating and -Nondefoliating Isolates of VerticiUium dahliae ~

Catalina Pgrez-Lara, 1 Encarnaci6n Pgrez-Art6s I and Rafael M. Jim6nez-Dfaz 1'2 1Dept. de Proteccidn de Cultivos, Instituto de Agricultura Sostenible, CSIC,

14080 Cdrdoba [Telefax: +34-57-218563]; and 2Dept. de Agronomth, Escuela T~cnica Superior de Ingenieros Agrdnomos y de Montes,

Universidad de Cdrdoba, 14080 Cdrdoba, Spain

Verticillium wilt of cotton (Gossypium hirsutum), caused by Verticillium dahliae, is widespread in the Guadalquivir Valley of southern Spain. Isolates of the pathogen can be characterized as highly virulent cotton-defoliating or mildly virulent cotton-nondefoliating by means of biological pathotyping. The cotton-defoliating pathotype, which has been shown to be lethal to susceptible olive (Olea europaea), is spreading to neighboring olive-growing areas. Biological pathotyping of isolates of V. dahliae is laborious, time-consuming, and may be influenced by the environment. Development of an improved method for isolate characterization would be of practical value for research on the epidemiology of, and resistance to, the disease. DNA from 11 cotton-defoliating and 15 cotton-nondefoliating isolates of V. dahliae from the Guadalquivir Valley was extracted and amplified by the polymerase chain reaction using 10-mer commercial oligonucleotides as random primers and a range of reaction conditions. The amplification products were analyzed by electrophoresis for polymorphisms to determine whether pathotypes could be distinguished at the molecular level. DNA polymorphisms obtained with some of the random primers correlated well with the pathotypes, suggesting that the RAPD technique may be valuable for the characterization of isolates of V. dahliae. (P)

Physiological, Pathological and Molecular Characterization of Verticillium albo-atrum Strains Isolated from Potato and Alfalfa

Tharcisse Barasubiye, Claude Richard, Daniel Dostaler, Jean-Guy Parent, Richard C. Hamelin and Serge Laberge

Agriculture and Agri-Food Canada, Research Station, Sainte-Foy, Quebec G I V 2J3, Canada [Telefax: +1-418-648-2402]

Pathogenicity and growth of strains of Verticillium albo-atrum recovered from alfalfa (Medicago sativa) and potato (Solanum tuberosum) were determined. On alfalfa, the alfalfa strains were more virulent than the potato strains; only the potato strains were virulent on potato. The optimum temperature for growth was 20~ for potato strains and 20 to 25~ for alfalfa strains on potato dextrose agar. No alfalfa strain was killed at 30~ whereas this temperature was lethal for 75% of the potato strains. On Fries medium, the potato strains produced three to four times more dry weight than the alfalfa strains. However, on a solid medium amended with potassium chlorate, a significant (P<0.05) inhibition was noted for potato strains as compared with alfalfa strains. These results indicate that V. albo-atrum strains isolated from alfalfa and potato form two pathologically and physiologically distinct groups.

o Paper not presented at the Symposium.

Phytoparasitica 23:1, 1995 41

Two molecular methods were used to study genetic variability within the strains: restriction fragment length polymorphism using mitochondrial DNA, and random amplified polymorphic DNA (RAPD) using total DNA. Little mitochondrial DNA polymorphism was detected between V. albo-atrum strains from alfalfa and potato. In contrast, RAPD patterns differed clearly between the alfalfa and the potato strains. Four RAPD markers were present in all of the alfalfa strains but absent in the potato strains. A significant (37%; P<0.002) portion of the total genetic variance was attributable to differences between the potato and the alfalfa strains. The results suggest that these alfalfa and potato strains are genetically, distinct. Furthermore, a fairly high level (63% of the total variance) of intra-group diversity was observed within strains from potato and alfalfa. In addition, two avirulent potato strains were clearly divergent according to RAPD patterns. These results, combined with those of previous inoculation and growth studies, are in agreement with the presence of host-specific strains in V. albo-atrum. (P)

Characteristics of English Isolates of Verticillium dahliae

D.C. Harris and J.R. Yang Horticulture Research International, East Mailing, West Malling, Kent

ME19 6B J, UK [Telefax: +44-732-849067]

Studies of restriction fragment length polymorphisms (RFLPs) in a collection of isolates of Verticillium dahliae have shown that two distinct types predominate among English isolates. Representatives of the two types were examined for vegetative compatibility (using the nit [nitrate nonutilizing] mutant system) and their pathogenicity. Twenty-four of the 43 isolates produced complementary nit mutants and were of two compatibility types which corresponded to the two RFLP types. When inoculated to a complex hybrid of Fragaria vesca X F. chiloensis X F. virginiana (clone UC5), the pathogenicity of the isolates varied: the mean pathogenicity of isolates from the two RFLP groups was different although the ranges of pathogenicity of isolates in the two groups overlapped. When a selection of these isolates was inoculated to the Fragaria hybrid, Fragaria X ananassa cv. Hapil, potato cv. Home Guard, and seedlings of Acer platanoides, the ranking of isolates for pathogenicity was consistent on the four hosts, indicating that there is no host adaptation in the two RFLP types. (L)

Diploid Isolates of VerticiUium dahliae, viz~ V. dahliae var. longisporum Stark Causing Vascular Wilt Disease in Oilseed Rape

V.K. Karapapa, B.W. Bainbridge and J.B. Heale Microbial Physiology Research Group, King's College, Kensington,

London W8 7AH, UK [Telefax: +44-71-333-4500]

Verticillium wilt of oilseed rape is caused by Verticillium dahliae var. longisporum Stark, a pathogen producing severe losses in Sweden, Germany, France and Poland. It has not been reported from the UK, but a potential threat exists in view of the very large area of the crop and the susceptibility of oilseed rape to diploid isolates of V. dahliae now present in the near continent. Inoculation tests involving various isolates of V. dahliae from different hosts and locations against five different cultivars of oilseed rape, showed haploid isolates to be non-pathogenic. A cold hydrolysis technique for Feulgen DNA microdensitometry indicated that all the pathogenic isolates to oilseed rape were diploid; DNA values for diploid pathogenic isolates were almost double the DNA values of haploid, non-pathogenic isolates.

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The distinct nature of this host-specific strain of V. dahliae was further investigated by RAPD (random amplified potymorphic DNA) analysis and by PFGE (pulsed-field gel electrophoresis) to detect chromosome length polymorphisms. Based on our work of more than 10 years with these diploid isolates, and particularly on their highly distinctive characteristics which separate them clearly from the related species V. dahliae, V. albo-atrum and V. tricorpus (stable diploidy, large conidia, irregular/elongated microsclerotia, lack of polyphenol oxidase activity with tannic acid as substrate, RAPD fingerprint and PFGE karyotype), combined with pathogenicity to a range of cruciferous hosts including oilseed rape, Arabidopsis, Chinese cabbage and Japanese radish - we propose that V. dahliae var. longisporum Stark be renamed as a new species of Verticillium, viz. Verticillium cruciferarum sp. nov. (P)

Molecular Studies and Relationships between Isolates of Verticillium dahlia and V. albo-atrum

D.J. Barbara, J.H. Carder, A. Morton and A.M. Tabrett Plant Pathology and Weed Science, Horticulture Research International,

East Mailing, West Mailing, Kent ME19 6B J, UK [Telefax: +44-732-849067]

Initial restriction fragment length polymorphism (RFLP) studies of isolates of Verticillium dahliae and V. albo-atrum, largely from the UK, showed that both species could be divided into clear groups, with little variation within the groups. Polymerase chain reaction primers which can identify some of the groups have been developed. Further RFLP studies have suggested that these groups may not represent the range of variation present outside the UK.

Long repeat sequences have been found in the intergenic regions of both species; the presence of such repeats is unusual for non-oomycetous fungi. The structures of the sub-repeat regions are distinct in three groups of isolates, v/z., haploid isolates of V. dahliae, and diploid isolates of V. dahliae and V. albo-atrum. Significant differences between isolates in the number of sub-repeats were found only within the group of V. dahliae haploid isolates; these differences were not correlated with the RFLP group.

Sequence differences within the internal transcribed spacers of the rRNA genes suggested some unexpected relationships. For example, diploid isolates of V. dahliae appeared to be closely related to V. albo-atrum and more distantly related to haploid V. dahliae isolates than were lucerne isolates of V. albo-atrum.

The significance of these results for the taxonomy of the two species was discussed. (L)

Variability in Virulence among Pathotypes of Verticillium dahliae and Possible Implications in IPM Programs for Potato Early Dying

Randall C. Rowe and Despina D. Botseas Dept. of Plant Pathology, OARDC, The Ohio State University,

Wooster, OH 44691, USA [Telefax: +1-216-263-3841]

Potato early dying (PED) is an important disease of potato (Solanum tuberosum) in temperate climates worldwide, including both the United States and Israel. Verticillium dahliae (Vd) is the primary pathogen involved in PED, although various other pathogens have been associated with the disease, especially the root-lesion nematode Pratylenchus penetrans (Pp). Pp interacts with Vd on potato, and co-infection results in higher disease severity and often lower yields than would occur with fungal infection alone. By testing complementation of nitrate non-utilizing mutants,


several vegetative compatibility groups have been shown to exist within populations of Vd and some relationship of these groups with virulence to potato has been demonstrated. Field microplot studies were conducted in Ohio with VCG 4A and 4B isolates of Vd to evaluate virulence to potato and ability to interact with Pp. No differences in virulence were found between isolates from VCG 4A and 4B when plants were grown in soil infested with Vd alone. In contrast, plants grown in soil infested with VCG 4A isolates of Vd plus Pp exhibited higher disease severity and had lower tuber yields, than plants grown in soil infested with VCG 4B plus Pp. These data indicate that isolates of Vd from VCG 4A may be able to interact synergistically witti Pp, whereas isolates from VCG 4B do not interact with Pp or do so only weakly.

It is likely that mixtures of several VCGs of Vd exist in many soils. These may vary in virulence on specific host plants and also in their ability to interact synergistically with root-lesion nematodes. Mixed populations of Vd may affect the use in yield-loss predictive models of microsclerotial population data obtained from soil assays and may partially explain inconsistencies" among reported economic threshold values for Vd in soil. (L)

Vegetative Compatibility Groups in Japanese Isolates of Verticillium dahliae Using Nitrate-Nonutilizing Mutants

H. Nagao, 1 D. Wakatabe, 1 T. Shiraishi 2 and T. I i j ima 3 1Laboratory of Plant Pathology, Faculty of Horticulture, Chiba University,

Matsudo, Chiba 271 [Telefax: +81-473-662234]; 2Gunma Agricultural Research Center, Gunma 379-22; and 3Tokyo Metropolitan Agricultural Experiment Station, Tokyo 180, Japan

In Japan, isolates of VerticilIium dahliae have been classified into six groups (or pathotypes) using a set of five differential hosts (Hagiwara, 1990). We tried to distinguish Japanese isolates using microsclerotial color mutants but could not obtain a clear result. Joaquim and Rowe (1990, 1991) used nitrate-nonutilizing (nit) mutants to reassess vegetative compatibility groups (VCGs) in V. dahliae isolates previously defined by Puhalla and Hummel (1981). In this study we examined vegetative compatibility relationships using nit mutants among 70 isolates of V. dahliae collected in Japan.

nit mutants were induced on minimal agar medium (MM) containing 3.0% potassium chlorate (MMC) for 14-21 days at 25~ Two complementary nit mutants, nit-1 and nit-M, were paired with all combinations on MM at 25~ for 20 days. The criteria for vegetative compatibility .were determined by the prototrophic growth between nit-1 and nit-M. A heterokaryon zone >5 mm wide was regarded as a positive reaction (++), a thin line (<5 mm) as (+), limited reactions as (-), and no reaction as (N).

Three main groups were recognized, corresponding to three pathotypes reported previously. We expect grouping will be standardized in the future, so we temporarily designated the three groups as VCGJ1, VCGJ2 and VCGJ3, respectively. VCGJ1 was compatible with both VCGJ2 and VCGJ3, but V(?GJ2 and VCGJ3 were incompatible with each other. VCGJ3 and VCGJ1 corresponded to eggplant pathotype and pepper pathotype, respectively. VCGJ2 was composed of isolates virulent to tomato. V. dahliae isolates of Race 2 for tomato were examined and shown to belong to VCGJ2. A newly found isolate that was pathogenic to both tomato and pepper was compatible with VCGJ1 and VCGJ3. Although this isolate was pathogenic to tomato, it was not compatible with VCGJ2. The isolates of soybean pathotype and weakly virulent isolates were described.

There seems to be substantial VCG diversity in Japanese isolates of V. dahliae, and vegetative compatibility analysis was a useful tool for classifying the different strains. (L)

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Use of DNA Hybridization and Vegetative Compatibility Groups for the Detection and Differentiation of Verticillium Species

E.J. Paplomatas 1 and K. Elena 1'2 1Benaki Phytopathological Institute, 145 61 Kifissia-Athens [Telefax: +30-1-807-7506]; and 2National Foundation of Agricultural Research, 151 25 Amaroussion-Athens, Greece

Pathogenicity of Verticillium dahliae isolates is generally nonspecific and formae speciales have never been found in this species. Although V. dahliae is a common soilborne plant pathogen in Greece, causing severe wilt diseases of a large number of annual crops and fruit trees, the genetic diversity of the Greek populations has never been studied. Furthermore, there is a need for a rapid and reliable method for the detection and quantification of the fungal biomass in the plant tissue and the soil, and its differentiation from V. tricorpus, another microsclerotial soilborne species. The goal of this study was to investigate the genetic relatedness of V. dahliae isolates through vegetative compatibility groups (VCGs) analysis and to develop specific DNA probes that would detect and differentiate Verticillium species. To obtain nit mutants, V. dahliae and V. tricorpus isolates were transferred from PDA to a minimal agar medium (MM) amended with KC103 (initially at concentrations of 15 g/l and later at 25 g//) and 1.6 g/l L-asparagine. The nit phenotypes of the chlorate-resistant mutants were determined by their ability to use nitrate, nitrite and hypoxanthine as a nitrogen source. Although relatively large numbers of chlorate-resistant mutants were obtained at the higher KC103 concentration (25 g/l), they were all characterized as nit-1 or nit-3. In complementation tests, these mutants were not able to form heterokaryons, which was rather expected, since complementation is rare between nit-1 and nit-3 phenotypes but much more frequent between nit-1 or nit-1 and nit-M mutants. Currently, a larger number of nit mutants are phenotypically characterized and new mutants are being developed at higher KC103 concentrations. For the production of random DNA probes, total genomic DNA from isolate 65 of V. dahliae was digested with EcoRI, ligated into phagemid pBluescriptlI KS+ also linearized with EcoRi, and used to transform competent ceils of Escherichia coli strain JM101. Transformants were selected based on blue/white color of E. coli colonies and insert size was determined after isolation of recombinant plasmids and digestion with EcoRI. A partial library of V. dahliae clones was obtained with insert sizes ranging from approximately 0.3 to 5.0 kb. To identify clones that could be used as probes suitable to reveal Restriction Fragment Length Polymorphism (RFLP), total genomic DNA from several V. dahliae isolates was digested with EcoRI and the resulting fragments were size-fractionated on 0.7% agarose TBE gels. The DNAs were transferred to nylon membranes and probed with nonradioactive labelled cloned DNA. Research is under way to further identify clones with inserts that would hybridize with repetitive sequences of V. dahliae genomic DNA to ensure the polymorphism necessary to differentiate the V. dahliae isolates. (L)


Study of Vegetative Compatibility in Verticillium dahliae ~

Ljudmila G. Portenko, 1 G.I. Akimov 2 and Veronika G. Poshnagova 2 1All-Russia Institute of Rapeseed Research, Russian Academy of

Agricultural Sciences, Lipetsk 398037, Russia; and 2Dept. of Cotton Genetics, Tajikistan Academy of Sciences, Dushanbe, Tajikistan

The objectives of our investigation were: (a) to study vegetative compatibility (VC) of 24 strains of Verticillium dahliae isolated from cotton and soil in several regions of Middle Asia and differing in virulence from cotton cultivars (races 0,1,2,3,4); and (b) to test the Middle Asia collection of strains for VC with 33 strains from several different host species (cotton, tomato, eggplant, pepper, strawberry, cucumber, okra) and nine different countries (Tajikistan, Russia, Moldova, Israel, USA, UK, France, Spain, Morocco). Nitrate-nonutilizing (nit) mutants were used in tests for VC. Complementation tests showed that all the strains of the Middle Asia collection belonged to a single VC group (VCG). A diversity in virulence within a single VCG indicates that new races of cotton wilt causal agent may arise as a result of the parasexual cycle. This group of the strains was tested for VC with the two strains T-9 and SS-4 from cotton, USA, previously assigned to VCG1 and VCG2, respectively (Puhalla and Hummel [Phytopathology 73:1305-1308, 1983]; Joaquim and Rowe [Phytopathology 80:1160-1166, 1980]). These 24 strains were shown to belong to VCG1. Complementation tests between nit mutants derived from 33 additional strains revealed three VCGs. Distribution of the tested strains among the three VCGs was as follows: 31, including T-9, in VCG1; one (SS-4) in VCG2; and one (strain 345 from strawberry, England) in a separate VCG. Self-incompatible strains were not revealed. Electrophoretic phenotypes of 26 strains of three VCGs were obtained by polyacrylamide gel electrophoresis of mycelial extracts. Five banding patterns of non-specific esterases were revealed: three for 24 strains of VCG1, one for strain SS-4, and one for strain 345. We gratefully acknowledge the help of A.P. Kharkova (Moldova), J. Katan (Israel), J.E. DeVay (USA), D.C. Harris (UK) and C. Boisson (France) in providing certain strains of V. dahliae used in this study. (L)

Vegetative Compatibility and Pathogenicity Groupings within VerticiUium albo-atrum

M. Rataj-Guranowska, 1 I. Walkowiak, 1 J.H. Carder 2 and D.J. Barbara 2 1Institute of Plant Genetics, Polish Academy of Sciences, 60-479 Poznan, Poland

[Telefax: +48-61-221122]; and 2Horticulture Research International, East Malling, West Malling, Kent ME19 6B J, UK

Isolates of Verticillium albo-atrum were tested for vegetative compatibility using pairs of nitrate-nonutilizing (nit) mutants. The 16 isolates from hop have been shown previously to be from one restriction fragment length polymorphism (RFLP) group (NL) and were assigned to four pathogenicity groups based on the reaction of hop cultivars; the two isolates from lucerne were from RFLP group L. Mutants were derived from 17 of the isolates (1974 was the exception); for each isolate, mutants with one or more of the phenotypes nit-l, nit-3 and nit-M were produced.

Nine isolates appeared to be self-incompatible. Eight isolates were shown to be self-compatible but, unfortunately, most mutants of four of these reverted to prototrophy. Of the four isolates which could be fully tested for vegetative compatibility grouping (VCG), two (1788 and 621) from the


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same hop pathogenicity group were placed in VCG 01. Isolate 451 from a distinct pathogenicity group, and isolate SW from lucerne, were placed in separate VCGs (02 and 03, respectively).

Preliminary results suggest that hop isolate 527 is compatible with isolate SW. This is surprising, as SW is from lucerne (i.e., in RFLP group L) and earlier results from other workers indicate that isolates from this host form a distinct VCG; further work is needed to clarify this matter. (P)

Verticillium Wilt of Potatoes: Causal Agents and Pathogen Detection

H.W. (Bud) Platt, 1 J. Robb, 2 R. Nazar 2 and X. Hu 2 1Agriculture Canada, Research Station, Charlottetown, P.E.I., CIA 7M8

[Telefax: +1-902-566-6821]; and 2Dept. of Molecular Biology and Genetics, University of Guelph, Guelph, Ontario NIG 2W1, Canada

Verticillium wilt of potatoes occurs in almost all potato production areas. Both Verticillium albo-atrum and V. dahliae are traditionally recognized as the causal agents of this wilt and these two fungi have also been implicated in the potato early dying or premature senescence disease of potatoes. Areas with a warm, dry climate and 'light' soils such as central Canada and U.S.A. are often associated with V. dahliae as the principal causal agent. V. albo-atrum predominates in wet, cool and/or 'heavy' soil conditions such as eastern Canada and U.S.A. In recent studies, isolates of V. tricorpus from the Netherlands and Prince Edward Island have been shown to cause potato wilt symptoms. Traditional disease monitoring, assessment and forecasting activities involve the use of selective media and species identification based on microsclerotial/dark mycelium presence. However, the occurrence of common morphological traits among these three species can affect accuracy of identification.

Studies of serological techniques for accurate and rapid identifications were unsuccessful (false positives and negatives) due to interference by serologically active aspects of these species and others often associated with potato plant and soil systems. However, the development of a polymerase chain reaction-based assay was found to distinguish accurately the presence of these three species in both plant tissues and soil. On-going investigations on techniques for quantification with this assay method have demonstrated some success. (P)

B: PHENOLOGY AND EPIDEMIOLOGY

The Threat Posed to Wilt-Susceptible Crops in the United Kingdom by Linseed Cultivation

D.C. Harris 1 and B.D.L. Fitt 2 1 Horticulture Research International, East Mailing, West Malling,

Kent ME19 6BJ [Telefax: +44-732-849067]; and 2 Rothamsted Experimental Station, Institute of Arable Crops Research, Harpenden, Herts. AL5 2JQ, UK

The cultivation of linseed (Linum usitatissimum) in the U.K. has increased substantially in the last decade, mainly in response to European Community subsidies designed to encourage the production of oilseed for non-food uses. There is mounting evidence that infection of linseed crops by Verticillium dahliae is common in the U.K., although the effects of the fungus on seed yield


have not been evaluated. High levels of soil infestation by V. dahliae have been found following linseed, indicating that this plant can act as an effective amplifier of soil inoculum. There is also some evidence that the disease is seedborne in this crop. The use of linseed in rotation therefore poses a threat to the production of wilt-susceptible crops such as potatoes, soft fruits and hardy ornamentals. (P)

Use of Isolated Cells to Detect Phytotoxic Activity of Verticillium dahliae Culture Extracts on Solanum melongena

I.S. Melo and J.K. Brar Centro Nacional de Pesquisa de Monitoramento e Avaliar de Impacto Ambiental,

CNPMA/EMBRAPA, 13820 Jaguariuna-SP, Brazil [Telefax: + 55-192-672202]

Verticillium wilt, which is caused by Verticillium dahliae, is one of the most destructive diseases of eggplant in Brazil. Screening tests for resistance to the pathogen were carded out under greenhouse conditions through inoculation with a spore inoculum of root cuttings. Four introductions (RV-I, RV-II, RV-III and RV-IV) showed high resistance. The commercial cultivars Embu, Florida Market and Campineira were highly susceptible to V. dahliae.

,Cell suspension cultures obtained from the callus of susceptible and resistant cultivars of eggplant exhibited differential cell death responses when incubated with culture filtrates from isolate VD-02 of V. dahliae. More cell death was verified in cells of the susceptible cvs. Florida Market and Embu than in cells from the tolerant cvs. RV-III and B-27, as assessed by staining with fluorescein diacetate. When inoculated with conidia of V. dahliae, cvs. Florida Market and Embu evinced chlorosis, necrosis and vascular browning 7 days after treatment. These two cultivars showed an average disease rating of 2.6 and 2.3, respectively, and cultivars RV-III and B-27 had ratings of 1.0 and 1.3, respectively. The action of the culture filtrate closely reflected the action of the isolate when inoculated in plants under greenhouse conditions. (P)

Survival of Verticillium albo-atrum in Alfalfa Seeds

H.C. Huang, G.C. Kozub and E.G. Kokko Agriculture and Agri-Food Canada, Research Station, Lethbridge,

Alberta TIJ 4B1, Canada [Telefax: +1-403-382-3156]

Hard seeds of alfalfa (Medicago sativa) were artificially inoculated with Verticillium albo- atrum Reinke & Berthold and assessed for survival of the pathogen under laboratory and field conditions. When infected seeds were stored air-dry, V. albo-atrum survival was low (less than 10% after 10 months) at above-freezing temperatures (20 and 30~ but high (more than 90% at 10 months) at sub-freezing temperatures (-20 and -10~ Survival of the pathogen was also low (10% at 3 months) in seeds buried in soil at 20~ but high (90% at 10 months) at -5~ More than 75% of the hard seeds remained viable after 18 months of air-dry storage at -20, -10, 4, 20 or 30~ The incidence of Verticillium wilt in alfalfa plants due to seedbome V. albo-atrum decreased with seed storage period, averaging 40% after seed was stored for 1 month and zero when seed was stored for 12 or 18 months. In three field experiments, survival of seedborne V. albo-atrum decreased with increasing period of burial, but at different rates in each year. The percentage of seeds with viable V. albo-atrum was greater when the seeds were buried at 10 cm compared with 5

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and 2 cm (48% vs 39% and 38%, respectively). The complete loss of viability of V. albo-atrum in infected seeds stored at 30~ for 6 months indicates that seedborne V. albo-atrum could be eliminated by storing alfalfa seeds at 30~ for one year. (L)

Arabidopsis thaliana as an Experimental Host of Haploid Isolates of Verticillium dahliae

A.M. Tabrett, 1 J.H. Carder, 1 S. Coomber 2 and D.J. Barbara 1 1 Plant Pathology and Weed Science, Horticulture Research International,

East Mailing, West Mailing, Kent ME19 6BJ [Telefax: +44-732-849067]; and 2 King's College, University of London, London WCIE 7HX, UK.

Diploid isolates of Verticillium dahliae are primarily pathogens of cruciferous plants and, conversely, these plants are rarely naturally infected by haploid isolates. We have shown that the cruciferous weed Arabidopsis thaliana can be readily infected experimentally by haploid isolates of V. dahliae (and V. albo-atrum). Arabidopsis has several advantages as a model for the study of the interactions between haploid isolates of V. dahliae and their hosts, not least that it has been studied intensively from the molecular standpoint. Some of our work with this host/pathogen combination is described. (P)

Influence of Crop Rotation on the Population of Defoliating and Nondefoliating Pathotypes of VerticiUium dahliae in Field Soils

J. Bejarano-Alcazar, 1 M.A. Blanco-Lopez, 2 J.M. Melero-Vara 3 and R.M. Jirn6nez-Dfaz 2'3

1Dept. of Plant Protection, CIDA Cdrdoba, 14080 Cdrdoba [Telefax: +34-57-202721]; 2Dept. of Agronomy, College of Agriculture, University of Cdrdoba,

14080 Cdrdoba; and 3Inst. of Sustainable Agriculture, CSIC, 14080 Cdrdoba, Spain

A population of Verticillium dahliae in clay soil of four fields (A-D) in the Lower Guadalquivir Valley of southern Spain was investigated using an Andersen sampler during the years 1985-89 (fields A and B) and 1988-91 (fields C and D). Cotton crops in the four fields were affected by Verticillium wilt in previous years. Field soils were infected with the cotton-defoliating (fields B- D) or the cotton-nondefoliating (field A) pathotypes of V. dahliae with initial inoculum density (ID) in the upper 20-cm soil layer ranging from 4.0-90.2 propagules (p)/g among fields. Crop rotation during the study included cotton, maize, sunflower or wheat, depending upon the field. In addition, several plots were solarized in fields C and D in the summer of 1988.

Initial ID in fields A and B was 10 and 4 p/g, respectively. However, population dynamics of the pathogen over time differed in the two fields. In field A, ID remained stable during the first 3 years of the study and thereafter increased up to 27.5 and 91.5 pig in 1988 and 1989, respectively. On the other hand, ID in field B increased up to 51 p/g after 2 years (1987) and reached 120 pig 2 years later (1989). ID in fields C and D increased from 39.5 and 64.0 pig in 1988 to 84.1 and 85.5 pig in 1990, and remained at the same level in 1991. Soil solarization in 1988 eradicated the pathogen from the soil to undetectable levels. Thereafter, ID increased continuously during the subsequent 3 years, reaching.27.0 affd 13.7 pig in fields C and D, respectively, in 1991. Fields infected with the defoliating pathotype of V. dahliae had a high incidence and severity of Verticillium wilt, and consequently contributed to increased incorporation of infected crop debris in the soil. (P)


Verticillium dahliae Incidence in Potatoes Grown after a Monoculture of Onions, Forage Maize, Flax,

Kidney Beans, Peas or Field Beans

J.G. Lamers Research Station for Arable Farming and Field Production of Vegetables,

8200 AK Lelystad, the Netherlands [Telefax: +31-3200-304791

Potatoes were grown after 10 and 11 years of continuous cropping of onions (Allium cepa), maize (Zea mays), flax (Linum usitatissimum), kidney beans (Phaseolus vulgaris), peas (Pisum sativum) and field beans (Vicia faba) on a calcareous loam polder soil. In these 2 years, Verticillium dahliae incidence was assessed approximately 10 weeks after planting by plating 50-60 basal stem cuttings on NPX-agar, and approximately 20 weeks after planting by examining 100 stems for the presence of microsclerotia. Also other diseases were assessed and the growth and yield of potatoes were determined.

In both years potatoes preceded by field beans showed significantly increased V. dahliae disease ratings (to a maximum of 84%). Only in the first year could a yield reduction of 10% be attributed to Verticillium disease. After peas, the Verticillium incidence increased significantly only 10 weeks after planting during the first year (to 44%) and 20 weeks after planting during the second year (to 36%). This occasionally increased Verticillium incidence was not associated with yield reduction. Onions, maize, flax, and kidney beans showed no or reduced Verticillium disease ratings following potatoes, although yield differences amounted to 15%. It is concluded that yield of potatoes differed according to the preceding crops as a consequence of adverse soil structure conditions (maize and onions) and V. dahliae (fielffbeans). (L)

Verticillium dahliae Infection of Potato Plants under Stress Conditions

Jana Orenstein, S. Sabbah and A. Nachmias Dept. of Plant Pathology, ARO, Gilat Regional Experimental Station,

M.P. Negev 2, 85280, Israel [Telefax: +972-7-926337]

Potato, originally bred in the temperate climate of northem Europe, is an important crop in semi-arid zones. It is moderately sensitive to salinity and drought, and susceptible to diseases such as Verticillium wilt. These conditions are the major limiting factors to potato production in hot climates.

The aim of the present experiment was to investigate the effect of salt (50 mM NaC1, which is equivalent to EC=10.2 mMho) and of drought (created by 10% polyethylene glycol) on potato growth and Verticillium infection. The work was conducted in the presence of the phytohormones kinetin and abscisic acid. A Verticillium dahliae spore suspension was added to the test tubes 2-3 days after planting. Sterile plantlets of potato cv. 'Nicola' were held in an artificial medium (vermiculite:perlite, 15:85) in a test tube.

Growth and pathogen colonization were determined. Salinity and drought stresses restricted growth and enhanced pathogen establishment in the plantlets, as measured by the number of colony-forming units (cfu). Abscisic acid increased the number of cfu more than kinetin, compared with control plantlets. (P)

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Enhancement of VerticiUium Infection in Potato under Stress Conditions

A. Nachmias, 1 L. Livescu, 1 Leah Tsror (Lahkim), 1

G. Ben-Hador, 1 Jana Orenstein, 1 S. Sabbah 1 and D. Orion 2 1Dept. of Plant Pathology, ARO, Gilat Regional Experimental Station, M.P.

Negev 2, 85280 [Telefax: +972-7-926337]; and 2Dept. of Nematology, ARO, The Volcani Center, Bet Dagan 50250, Israel

Verticillium wilt caused by Verticillium dahliae is the major fungal disease of potatoes growing in hot climates. In the Negev Desert potatoes are exposed to several stresses, such as: extreme heat, drought or temporary drought conditions, salinity, short days during winter, and nematode infection. Potato production under these conditions can be successful through the use of several agrotechnlcal measures that include: soil preparation, advanced irrigation systems, and careful pest and disease management. However, when VerticiUium infection occurs, yield reduction is inevitable and cannot be prevented. This study demonstrates an increase in V. dahliae infection in potato under these stress conditions.

The experiments took place in the field and in situ. Saline conditions were brought about by providing sodium and calcium chlorides (4:1) through the irrigation systems at a final concentration of 5 mMho cm -1; drought conditions were achieved by reduction of irrigation water supplies down to 25% of the evaporation pan; and nematode infection was achieved by planting the experiment in Pratylenchus mediterraneus-infested soils. In all the experiments V. dahliae infection increased significantly, as measured by: increased pathogen population in xylem bundles, disease symptoms development, and potato yield reduction.

It is suggested that a common physiological mechanism is involved in the disease enhancement of potatoes growing under conditions of salinity, drought, short days, and nematode infection. (L)

Effects of Various Cultural Practices on the Epidemiology of Verticillium Wilt of Cotton in New South Wales, Australia

S.J. Allen Australia Cotton Research Institute, Narrabri, New South Wales,

Australia 2390 [Telefax: +61-67-931186]

Irrigated cotton production was introduced into Australia in the early 1960s and Verticitlium wilt (Verticillium dahliae Kleb.) was soon recognized as an important disease of the crop. A comparison of isolates of the pathogen from cotton in Australia with isolates from the U.S.A. has shown that the Australian isolates are similar to, though less virulent than, the mild, non-defoliating isolates of V. dahliae from the U.S.A. Recently, the Australian isolates were described as belonging to the VCG 4 group which cause mild to moderate symptoms, even in susceptible cotton cultivars. Despite the presence of only mild isolates of the pathogen, significant yield reductions occur.

Commercial cotton crops in all of the major cotton-growing areas of New South Wales have been surveyed annually since the 1984/85 season, in order to monitor the distribution, incidence and severity of cotton diseases.

The mean incidence of foliar symptoms of VerticiUium wilt of cotton increased from 4.1% in the 1984/85 season to 16.5% in the 1989/90 season. During the same period the mean incidence of the disease in the Namoi Valley increased from 4.7% to 30.1%. This rise coincided with the increased adoption of minimum or reduced tillage practices and permanent bed systems, which


have resulted in greater retention of crop residues from season to season. In addition, crop residues are spread from field to field in irrigation water. The incidence of Verticillium wilt increases rapidly when newly developed fields are irrigated with water which has run off a field where the disease is well established. The pathogen is easily isolated from crop residues in this re-circulated water. Repeated cotton cultivation in the same field year after year, and the use of susceptible cultivars, have also contributed to the increase in mean disease incidence.

The mean incidence of Verticillium wilt has decreased since reaching a peak in the 1989/90 season. A major factor in this decrease was the commercial release of the cultivar 'Sicala VI ' which has significant resistance to V. dahliae. New cultivars with still higher levels of resistance are soon to be released. A second factor which may have contributed to the decreasing incidence of the disease has been the widespread adoption of raking and burning of crop residues. The results of several replicated field experiments comparing raking and burning with stubble-mulching have failed to demonstrate a direct reduction in the incidence of Verticillium wilt in the fullowing season. However, the long-term effects of raking and burning crop residues at the end of the season have not been determined.

The results of annual disease surveys of individual fields demonstrate the importance of tillage systems, crop residue management, and cultivar in the epidemiology of Verticillium wilt of cotton in New South Wales. (L)

Verticillium Wilt of Cauliflower: A New Disease in California

S.T. Koike I and K.V. Subbarao 2 1 University of California, Cooperat&e Extension, Salinas, CA 93901

[Telefax: +1-408-758-3018]; and 2Dept. of Plant Pathology, University of California, Davis, c/o U.S. Agricultural Research Station, Salinas, CA 93905, USA

In 1990, commercial cauliflower plantings in the Salinas Valley showed symptoms of a vascular wilt disease. Symptoms consisted of chlorosis, defoliation, stunting, wilting, and vascular discoloration of the root and stem. Surveys indicated that the incidence of symptomatic plants in affected fields could be as high as 95%. This problem was initially detected in a few fields in two counties; today the disease is widely distributed in five coastal counties. VerticiUium dahliae (Kleb.) was consistently isolated from cauliflower stem tissue, and pathogenicity tests using root dip techniques confirmed that this fungus was the causal agent. Other crucifers grown in the Salinas Valley, such as cabbage and Chinese cabbage, are also susceptible to this pathogen. However, commercial broccoli fields remained unaffected, even though such crops were planted in highly infested soils. Soils from the Salinas Valley were assayed for microsclerotia by plating dried soil onto NP-10 selective media using a modified Anderson sampler. Propagute counts show that V. dahliae is widely distributed in the region, with populations as high as 93 microsclerotia/g soil. All commercially available cauliflower cultivars were found to be susceptible to the pathogen, although they varied in the degree of symptom severity. Soil fumigants (chloropicrin, chloropicrin + methyl bromide, Vapam [metham-sodium], Mocap [ethoprophos]) injected into pre-formed beds but left untarped failed to reduce soil inoculum or increase yields significantly. The ability of V. dahliae to survive in soil for long periods of time, the lack of resistant cuttivars, and the prohibitive costs of standard tarped soil fumigation applications make this disease a serious threat to the cauliflower industry in California. (L)

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C: MICROSCLEROTIA

The Production of Microsclerotia of Verticillium dahliae for Use in Studies of Survival

Mary Anne Hawke and George Lazarovits Agriculture Canada, London Research Centre, London, Ontario N5V 4T3,

Canada [Telefax: + 1-519-645-4085]

A procedure for the laboratory production of stable populations of microsclerotia (MS) of Verticillium dahliae, which can be kept for extended periods without loss of viability, is described. The fungus is grown in a semi-solid, modified Czapek-Dox medium for several weeks, after which it is blended and wet-sieved to isolate the MS from hyphae and conidia. The MS are mixed with acid-washed quartz sand so that they dry without clumping. They can be removed from the sand and separated into different sizes by dry sieving. MS viability is determined by transferring individual MS with a sterile needle to plates of soil-pectate-tergitol agar (SPT). A grid of 25 squares is placed under the plates and one MS is placed within each square. Survival is evaluated by counting the number of colonies after a minimum of 2 weeks' incubation at 24~ Examination of the effect of MS size on growth and survival showed that 75-106 ~tm MS exhibited faster, more synchronous germination and a faster rate of colony growth than MS either 53-75 or <53 ~tm in size. Larger MS generally survived long-term storage better and exhibited less variability in germination at 24~ than did smaller MS. Populations of 75-106 txm MS obtained by this procedure showed 94% germination after 59 weeks' storage at 24~ There was little difference in survival of MS when stored dry, in Eppendorf tubes, for 10 weeks at 24, 4, -25 or -70~ Storage in the freezer compartment of a frost-free refrigerator, however, was much more damaging to MS than storage in a chest-type freezer at -25~

The ability of the procedure to reflect both lethal and sub-lethal treatment effects was demonstrated by the results obtained following exposure of melanized and non-melanized MS to 254 nm UV irradiation. Germination of non-melanized MS was reduced by 50% after 2 h irradiation, and these MS were killed after >6 h exposure. There was only a slight reduction in the germination of melanized MS after >36 h of exposure; however, the colonies produced were less than half the size of those produced by melanized MS not exposed to UV light. In exhibiting reduced colony growth, the irradiated, melanized MS were behaving like smaller MS, which contain fewer cells. Measurement of colony diameter, therefore, can reveal sub-lethal treatment effects. In another assay for survival, nylon packets containing MS/sand mixtures were buried for 3 to 4 weeks in soils having differing inherent levels of microbial activity. After recovery and plating of the MS onto SPT, the colonies which grew were scored in two different ways: (i) as the presence or absence of a colony; or (ii) the colony was scored as being (a) V. dahliae, (b) V. dahliae mixed with another fungus inhibitory to its growth, (c) V. dahliae co-existing with another fungus, (d) a fungus which was other than V. dahliae, or (e) no colony of any kind formed. Scoring only the presence or absence of a colony revealed little information about the fate of the MS. When the data from the second scoring system were analyzed by the chi-square test, however, they clearly revealed differing patterns of activity (both lethal and sub-lethal) in the soils.

The usefulness of this procedure as a tool, and the ecological implications of this approach to studying the survival of MS of V. dahliae, were discussed. (L)


The Influence of Plant Roots on the Germination of Microsclerotia of VerticiUium dahliae in the Soil

L. Mol Dept. of Agronomy, Wageningen Agricultural University, 6709 RZ

Wageningen, the Netherlands [Telefax: +31-8370-84575]

A technique with root observation boxes was developed to study the interaction between microsclerotia (MS) and plant roots. Wooden boxes with one transparent side and non-sterilized soil were placed slantwise. After planting, roots grew on the inner side of the transparent wall on which MS were fixed in a thin water-agar layer. With this system it was possible to measure non- destructively: the percent of MS that germinated, and the number of hyphae per germinated MS in relation to the distance of the MS from the root surface. Counts and measurements were conducted in a radius of 1 mm around five root tips. Two experiments were carried out twice, with five replications. In the first one, four crops were grown: potato cv. Element (a susceptible and sensitive cultivar), potato cv. Mirka (a rather resistant and tolerant cultivar), barley, and field bean. In the second experiment seven crops were grown: potato cv. Element, potato cv. Ostara (an early cultivar), potato cv. Astarte (a late cultivar), pea, sugar beet, onion, and flax. In both experiments a box without a crop was used as the control. In the control, five spots with a radius of 1 mm were chosen at random. A field trial on a sandy soil was carried out to compare the root length of crops grown under the same conditions. The root length was estimated using the line-intersection method.

Roots of host crops had a stronger stimulating effect on germination of MS per root tip than crops that are known as non-hosts. Combining the results of the experiments using root observation boxes with field observations on root lengths, showed that crops with a relatively weak stimulating effect per root tip can give a greater reduction of viable MS in the soil than crops with a relatively strong stimulating effect per root tip, because of large differences between root densities of crops.

The chances of total elimination by spontaneous germination of MS may be low, because MS have the ability to germinate more than once. However, the experiments showed deafly that crops can contribute to a reduction of the inoculum density in the soil. Potato cultivars differed in their capacity to induce germination of MS, indicating that differences in susceptibility to V. dahliae infection among cultivars can be partly explained by differences in their capacity to induce germination of MS. (L)

The Role of Melanin in the Survival of Microsclerotia of Verticillium dahliae

Mary Ann Hawke and George Lazarovits Agriculture Canada, London Research Centre, London, Ontario N5V 4T3,

Canada [Telefax: +1-519-645-4085]

Melanins are brown to black pigments which are presumed to play an important role in the survival of fungal resting structures. In Verticillium dahliae, the melanin is a product of the biosynthesis of 1,8 dihydroxynaphthalene from pentaketide and is located within the cell walls and between the cells of the microsclerotia (MS). The role of melanin in V. dahliae was studied with the aid of an albino mutant (Aim, provided by Dr. M. Wheeler) or by the addition of tricyclazole (TCZ) to the growth medium at either 1 or 10 mg/l. TCZ, a systemic protectant used to control rice blast disease (Pyricularia oryzae), specifically inhibits the reductase of the pentaketide melanin

54 Meetings

pathway. We studied the effects of blocking melanin biosynthesis on germination, growth and survival of MS of V. dahliae, in addition to the survival of MS exposed to various abiotic and biotic factors.

In both melanized MS and MS which had melanin production blocked by TCZ, the rate of MS germination was closely correlated with the size of the MS: 75-106 Ixm MS exhibited faster and more synchronous germination than did MS either 53-75 or <53 ~tm in size. In melanized MS, the rate of colony growth was also correlated with MS size and 75-106 txm MS grew faster than MS <75 Ixm. MS size and rate of colony growth were not correlated in MS treated with TCZ. When MS were stored at 24~ for up to 35 weeks, the melanized MS generally survived better than those receiving TCZ. In one experiment, both melanized and non-melanized MS still reached 100% germination after 35 weeks. In a second experiment, melanized MS attained 100% germination at times when MS grown with 1 or 10 mgll TCZ reached 80% and 42% germination, respectively.

The survival of melanized and non-melanized MS after exposure to abiotic factors, such as UV irradiation or storage temperature, was determined. When exposed to UV irradiation at 254 nm, all albino MS were killed after 6 h, whereas the germination of non-irradiated controls or melanized MS was virtually unaffected even after 48 h. Germination of MS in which melanin formation was blocked by 1 or 10 mg/l TCZ was only slightly affected by irradiation of >24 h, but the size of the colonies after 4 days' growth was greatly reduced compared with non-irradiated controls. These findings confirm the role of melanin as a protectant against damaging radiation. The presence of melanin, however, did not appear to influence the survival of MS of 75-106 ktm size when stored at 24, 4, -25 or -70~ for a short period of 10 weeks.

Exposure of melanized and non-melanized MS to biotic factors such as organic amendments or antagonistic organisms was examined by burying nylon packets containing mixtures of acid- washed quartz sand and 75-106 ~tm MS in soils under controlled conditions. Whereas the germination of melanized MS was >96% when buried 4 weeks in soil amended with 1% (w/w) chitosan, bloodmeal or a fishmeal/bonemeal/kelp mixture, the germination of MS receiving 1 or 10 mg/l TCZ was 64, 84 and 93% or 49, 77 and 92%, with the different amendments, respectively. When MS were buried for 20 or 35 days in soil which was sterilized and inoculated with one of several antagonistic fungi, survival of TCZ-treated MS was very low compared with melanized controls. (L)

The Recovery Rate of Microsclerotia of VerticiUium dahliae with Different Detection Methods

A.J. Termorshuizen Dept. of Phytopathology, Wageningen Agricultural University, 6700 EE


Methods for the detection of microsclerotia (MS) of Verticillium dahliae have been improved by changing medium composition or incubation conditions (see e.g. Harris et al., Pl. PathoL 42:238-250, 1993). However, laboratories tend to obtain different results using the same methods. These may be caused by differences in drying period of the medium, air humidity during incubation, water quality, etc. Another feature may be that one method is optimal for certain soil types, but not for others. For these reasons, comparisons of results between research workers are essentially difficult. Thus, the comment Powelson and Rowe (Annu. Rev. PhytopathoL 31:111-126, 1993) made on different population densities of V. dahliae among production areas may well be caused by the features addressed above.

Currently, I am developing a standard procedure which would act beyond the different methods used, aiming to determine the recovery rate of the method. If the recovery of a method is known,


results from different laboratories could be compared with less bias. The method is based on the germination percentage of MS from colonized potato stems collected from the field and added to the soil suspension or to the soil dilution plate directly. This germination percentage (GP1) is compared with that of sterile-water-washed MS added to agar plates without soil suspension (GP2). The new estimate for the soil MS density is the number of MS as determined by one of the agar plate methods multiplied by GP2/GP1. Additional checks are made by the addition of known numbers of MS to the soil. It appears that on ethanol agar (= 20 g l -l agar + 5 g 1-1 ethanol + 50 mg 1-1 oxytetracycline) the recovery ranges from 5-35 (-70)%. In a field experiment on a clayey soil, the recovery (ranging from 20-30%) showed a positive correlation with the temperature of the soil at the time of sampling.

In some experiments percentages of germination were extremely low (<1%) on the soil dilution plates (both of the MS added and of the MS present in the soil), although in the absence of soil suspension, washed MS germinated by more than 80%. We are not able to explain this phenomenon. MS mixed with the soil or the soil suspension may be damaged by sharp soil particles. However, the germination of MS from sterilized soil suspensions is the same as that in the absence of soil. Also, the residence time of washed MS present in the soil suspension may be important, although it does not influence the germination of washed MS in water suspension. Currently, I am testing the hypothesis that there is an interaction between residence time of MS in the soil suspension and the influence of microbial activity on germination of MS of V. dahliae. The method will also be applied to media which are recommended by Harris et al. (1993).

The point advanced by Harris et al. (1993: 248) that added MS may bear little relation to propagules occurring naturally in soil is, in my opinion, a valid statement. If the procedure is applied with the appropriate checks, the recovery obtained with the present method will not lead to overestimation of the soil population. Since the recovery method implies the estimation of two quantities (the classic inoculum density and recovery), the precision of the method (indicated by the standard deviation) will increase, but the accuracy (indicated by the mean) will approximate more closely the real density of MS in field soil. (L)

D: BIOCHEMISTRY AND PHYSIOLOGY

Cell-Wall-Degrading Enzymes and Toxins of Verticillium dahliae

H.C. Dube Dept. of Life Sciences, Bhavnagar University,

Bhavnagar 364 002, India [Telefax: +91-278-28422]

The ability of a pathogen to infect the host depends on its elaboration of chemicals, among which the cell-wall-degrading enzymes, especially the pectolytic enzymes, have received the most attention. The types of enzymes that break the glycosidic bond by different modes with varying efficiencies, conditioned by the nature of the substrate and fidelity for terminal or random cleavage, have been fully elucidated. These enzymes are indispensable wherever tissue is degraded and cells are separated. The regulation of their production and modulation of their activity by several ambient factors have a direct bearing on their role in disease causation.

We compared the roles of cellulases, hemicellulases and pectic enzymes produced by Verticillium dahliae (causing cotton and tomato wilts) and V. albo-atrum (causing rot of banana stalk). The effect of sugars, amino acids, metal ions and fungicides on their production and activity was examined. The effect of host and non-host cell walls on the induction of polysaccharidases of V. dahliae (tomato isolate) was studied. The cell-wall-degrading enzymes were produced

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sequentially on both host and non-host cell walls, the pectic enzyme being the first to appear, followed by the hemicellulases and cellulases. Pectic enzyme production was delayed by the non- host cell wall.

The reason for the ability to infect the host and not the non-host remains unclear. The freedom of the non-host from infection could be attributed, inter alia, to cell walls especially in view of the 'switching points' (sensu Heath, 1974) noted during invasion of non-hosts. There are reports on failure or reduced success of fungi to penetrate non-hosts, in spite of formation of infection structures (Politis, 1976). The influence of cell walls on the synthesis of polysaccharidases by pathogens, or on the activity of cell walls, may play an important role in determining resistance to invasion. While no significant differences in the pattern of enzyme secretion have been noted in the presence of susceptible or resistant plant cell walls (Jones et al., 1972), a distinct difference in the levels of synthesis of pectic enzymes has been reported (Cooper et al., 1981) in the presence of host and non-host cell walls. (L)

Role of Lectins in Verticillium Wilt of Cotton ~

L.N. Ten, 1 M.M. Faiziev 2 and L.A. Romanova 1 1 Tashkent State University and 2 Institute of Microbiology, Tashkent,

Republic of Uzbekistan

Specific proteins taking part in the processes of intercellular recognition and in inducing host defense reactions in the cotton-Verticillium wilt agent system were studied.

From a resistant cultivar of cotton, 'Mexicanum', and from the susceptible cv. C-4727, proteins with molecular mass 5000-45,000 D, agglutinating erythrocytes and spores of Verticillium dahliae were isolated. Lectins of these objects differ in agglutination titer, ability to agglutinate V. dahliae spores of races 1 and 2, carbon specificity and accumulating dynamics during vegetation.

Among proteins, accumulating in response to infection of cotton by V. dahliae, fractions indicating agglutinating ability were also determined. These lectins differ from the above- mentioned in molecular weight, carbohydrate and protein compositions. Velocity of these lectins syntheses was considerably higher in Mexicanum cotton compared with susceptible cv. C-4727 and a maximum of their accumulation precedes the formation of necrotic zones on leaves. Probably, a sensitizing action of one of the metabolites of V. dahliae - flaviolin, preliminary treatment by which at a concentration of 0.01% had no considerable effect on induction of phytoalexins (PA), led to increasing lectin levels in plants and is accompanied by a rapid increase of PA synthesis during pathogen penetration into the plant, brought forth by the de novo synthesis of lectins.

The obtained data point to the participation of cotton lectins both in pathogen recognition and in processes controlling PA synthesis. (P)


Phytoparas i t ica 23:1, 1995 57

E: RESISTANCE AND TOLERANCE OF PLANTS

Studies of the Resistance of Cocoa (Theobroma cacao) to Vascular Wilt Caused by Verticillium dahliae

Mario L.V. Resende, 1 Richard M. Cooper 2 and Julie Flood 2 1CEPLAC/CEPEC, C.P. 07, 45600-O00-ltabuna-BA, Brazil

[Telefax: +55-732-143200]; and 2School of Biology and Biochemistry, University of Bath, Bath BA2 7AY, Avon, UK

Verticillium wilt of cocoa has become an increasingly serious problem in Brazil, the world's second largest producer of this commodity. From the vast Amazon basin, the center of origin of the cocoa tree, approximately 22,000 accessions of Theobroma spp. have been collected and maintained by CEPLAC - Ministry of Agriculture in Brazil. However, the enormous variation within this population has not been adequately exploited in terms of resistance to diseases, mainly due to the lack of quick and efficient screening procedures. We are investigating the efficacy of methods for plant selection, pathogen variation and mechanisms of resistance to vascular wilt.

Sgil drench and stem puncture inoculation were compared as methods for selecting cocoa material, resistant to Verticillium dahliae. Disease progress was quicker following stem puncture than soil inoculation, and differences between cultivars under glasshouse conditions could be detected as early as 15 days after inoculation. Moreover, inoculum densities as low as 10 4

conidia/ml were sufficient to differentiate between resistant and susceptible cultivars by stem puncture, whereas with soil drench 107 conidia/ml were necessary. Also, the resistance of cultivar Pound-7 proved to be stable to very high inoculum densities (up to 108 conidia/ml), whichever method of inoculation was used. With either method, older seedlings were more susceptible to V. dahliae than younger ones. Nevertheless, due to the increased symptom expression with stem puncture, even 15-day-old seedlings presented a sufficient level of infection for assessment of resistance. In a nursery trial conducted in Brazil with seedlings at this age, seven cocoa genotypes previously selected as resistant, moderately resistant or susceptible to V. dahliae based on root inoculation, ranked in the same order when stem punctured. Stem puncture inoculation on young seedlings proved to be cost effective in terms of time and space and is therefore the best method to screen cocoa for wilt resistance, especially in a large-scale breeding program.

Studies were also carded out on pathogen variation between countries and regions, in terms of pathogenicity and genetic relatedness, as judged by vegetative compatibility using nitrate nonutilizing mutants. Isolates originating from cocoa in Colombia (VCG-4) and from cotton in the U.S.A. (VCG-1), caused severe defoliation on Pound-7, overcoming the resistance of this cultivar selected in Brazil. Cocoa isolates from different Brazilian states, even though in the same vegetative compatibility group (VCG-2); differed in their pathogenicity towards cocoa; this has provided critical information on the choice of isolates for selection trials. (L)

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The Role of Tannins and a Multiple Phytoalexin Response in the Resistance of Cocoa (Theobroma cacao) to VerticiUium dahliae

Mario L.V. Resende, 1 Richard M. Cooper, 2 Julie Flood 2 and Michael H. Beale 3 1CEPLAC/CEPEC, C.P. 07, 45600-O00-Itabuna-BA, Brazil

[Telefax: +55-732-143200]; 2School of Biology and Biochemistry, University of Bath, Bath BA2 7AY, Avon, UK; and 3Long Ashton Research Station,

University of Bristol, Bristol BS18 9AF, Avon, UK

Following the establishment of stem puncture as an efficient method to inoculate cocoa seedlings with Verticillium dahliae, this method is now used in the study of the biochemical mechanisms of resistance to Verticillium wilt in this crop. Based on evidence from the literature with other pathogens from cocoa, the level of phenolic compounds, including tannins, was initially investigated in susceptible and resistant cultivars, before and after inoculation. Despite levels of preformed condensed tannins in cocoa being toxic to spores of V. dahliae, increased concentrations were also found in three cultivars, 60 days post-inoculation. Nevertheless, no significant differences in tannin content were detected between susceptible and resistant cultivars. These results led us to consider other, untested methods to extract antifungal compounds from cocoa. Of four organic solvents with increasing polarities (petroleum ether, diethyl ether, ethyl acetate and methanol), diethyl ether was the most effective, extracting three highly inhibitory substances, here designated C-l, C-2 and C-3. Separation was achieved by flash chromatography combined with silica gel thin layer chromatography (TLC). These compounds were detected under UV light or by spraying the TLC plates with V. dahliae conidia. Mean Rf values with diethyl ether:petroleum ether:methanol (6:3:1) were, respectively: C1 = 0.46 and C3 = 0.25. Their structure is currently under investigation using nuclear magnetic resonance (NMR) and GC-MS spectroscopy. Compound-3 has already been identified as the triterpenoid arjunolic acid. The less polar substance (C-l) is an aliphatic long chain fatty-acid derivative. C-2 is 4- hydroxyacetophenone. Toxicities (as EDs0 ) to germination of V. dahliae conidia are (I.tg/ml) C-l, 3; C-2, 7; and C-3, 12; and levels in planta (~tg/g fresh weight) are 51, 2 and 168, respectively. EDs0 of tannins was found to be >400 gg/ml. Thus C-1 and C3 could contribute to resistance to V. dahliae. These substances, not present at detectable levels in intact noninoculated plants, can be considered as novel phytoalexins. Bioassays indicated that they accumulate only in the resistant cultivar Pound-7, not in a susceptible cultivar, and reach a maximum around 10 days after inoculation. We intend to evaluate their possible use as tools in breeding for resistance. (L)

Verticillium Colonization in Selected Resistant, Tolerant and Susceptible Alfalfa Plants as Determined by PCR-Based Assay

H. Xu, R.N. Nazar and J. Robb Dept. of Molecular Biology and Genetics, University of Guelph, Guelph, Ontario NIG 2W1, Canada [Telefax: +1-519-837-2075]

The development of Verticillium albo-atrum in alfalfa plants was studied by polymerase chain reaction (PCR)-based quantitative assays. Using stem infusion and root infection experiments, selected plants with known disease resistance were studied to determine colonization patterns by comparison of fungal biomass at different sampling times in various plant tissues (i.e., root, lower stem, upper stem and leaf). Results show that symptom expression is correlated closely with the levels of pathogen colonization or the amount of Verticillium present in the upper stem and leaf


tissues. Resistant plants of cvs. Vertus and Barrier prevent the development of wilt symptoms by confining the fungus to the lower stem and root tissues; such plants can harbor large amounts of V. albo-atrum in the lower stem and root. In contrast, susceptible plants of cvs. Vertus and Trumpetor show severe wilt symptoms and extensive colonization throughout.

We define tolerant plants as those expressing mild symptoms with no substantial effect on yield. Unexpectedly, tolerant plants of cvs. Barrier and Appollo were found to have substantially less pathogen in the lower parts of the plant at all sampling times than either their resistant or susceptible counterparts, but intermediate yet substantial amounts of fungus were detected in the upper stem portions. Clearly, the accurate monitoring of levels of Verticillium in symptomless and near-symptomless carrier plants is essential for effective disease management in this crop. (P)

Regulation of Resistance to Verticillium albo-atrum in Alfalfa

B.W. Pennypacker, D.P. Knievel, M.L. Risius and K.T. Leath Dept. of Agronomy, Pennsylvania State University, and USDA-ARS,

University Park, PA 16802, USA [Telefax: +1-814-863-2717]

Previously reported growth suppression in resistant alfalfa infected with Verticillium albo- atrum, concomitant with no reduction in net photosynthesis (Pn), alluded to the cost of resistance and to a regulatory role for Pn in the expression of resistance. Light affects the rate of Pn early in carbon assimilation by providing the energy needed to regenerate ribulose bisphosphate, the sugar substrate required by the carboxylase enzyme, Rubisco. For this reason, photosynthetic photon flux density (PPFD) was used to alter the rate of Pn to determine whether the rate of carbon assimilation regulates resistance to V. albo-atrum.

The experiment was a split-plot with a factorial treatment arrangement. Treatments were pathogen (V. albo-atrum and no V. albo-atrum), clone (resistant and susceptible), PPFD (100%, 70% and 40% of ambient), and time (3 weeks). Treatment effects were determined by disease ratings, dry weight analysis of plant parts, and measurement of Pn, stomatal conductance, dark respiration, in vivo and in vitro Rubisco activity, and quantity of Rubisco. Physiological parameters were determined on the resistant clone only. The experiment was repeated several times. Significant pathogen x PPFD x week interactions were deteeted in disease rating, plant height, stem dry weight, and aerial biomass, while pathogen x PPFD interactions were noted in leaf dry weight, dark respiration, and quantity of Rubisco when data from the resistant clone were analyzed. In all cases, interactions were caused by the loss of resistance in resistant plants grown under 40% PPFD. A significant pathogen x week interaction was noted in in vitro activity of Rubisco and indicated that infected, resistant plants had higher Rubisco activity than noninfected plants at all PPFD levels. V. albo-atrum did not affect Pn or stomatal conductance of the resistant clone; however, 40% and 70% PPFD significantly reduced both parameters. The significant effect of PPFI:r6n Pn and the lack of a pathogen effect, indicated that the amount of photosynthate accumulated by the infected plants, rather than the rate of net photosynthesis, was critical for the expression of resistance. The increased activity of Rubisco in the infected, resistant plants, and the decreased Rubisco activity previously reported in the susceptible plants, may be indicative of a resistance mechanism.

The susceptible clone was included in the growth parameter experiments to determine whether its response to PPFD was similar to that of the resistant clones. Inoculation of the susceptible clone differed by 6 weeks between the two experiments. Resistant and susceptible clones were treated identically in experiment 1, and the susceptible clone was inoculated 6 weeks later than the resistant clone in experiment 2. The infected, susceptible clone failed to respond differentially to the PPFD levels when treated identically to the resistant clone, which did respond to PPFD. The

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inability of the susceptible clone to alter its response to V. albo-atrum is evidence that the defense mechanism operating in the resistant clone is unique to the latter.

This research was supported by USDA NRICGP Postdoctoral Fellowship #91-37303-6434 from the Cooperative State Research Service, U.S. Department of Agriculture. (L)

Verticillium Wilt: Comparison of the Tolerance Phenomenon in Potato with Ve Gene Resistance in Tomato

Leah Tsror (Lahkim) and A. Nachmias Dept. of Plant Pathology, ARO, Gilat Experiment Station,

M.P. Negev 2, 85280 Israel [Telefax: +972-7-926337]

Intervarietal grafts have been inoculated with Verticillium dahliae to study the relative roles of roots and shoots in producing the vascular wilt syndrome in tomato and potato, When the resistant (RVF) tomato cultivar was used for rootstock experiments, the disease level of the graft was similar to that of the ungrafted resistant cultivar. Conversely, when the susceptible (RF) tomato cultivar served as rootstock, disease symptoms were the same level as those observed in the ungrafted susceptible plant. The results indicate that in tomato, the susceptibility of the grafted plant depends on the source of the rootstock. Moreover, the results suggest that the root serves as a major site of general resistance to Verticillium wilt in tomato.

In potato, grafting the susceptible 'Nicola' scion onto the highly tolerant 'Desir6e' rootstock reduced the post-graft level of colonization. When the susceptible Nicola served as rootstock, however, the number of colony-forming units above the grafting point had increased more than twofold over the pre-graft level 23 days after inoculation. The results seem to indicate that, as in tomato, Verticillium susceptibility lies in the rootstock. On the other hand, the results of root- dipping experiments suggested that a general tolerance mechanism may exist in potato as well, because, despite the low level of V. dahliae colonized in the tolerant cultivars, either no symptoms or only mild symptoms were observed.

In experiments designed to determine whether the Ve gene plays a role in Verticillium wilt tolerance in potato, races 1 and 2 of V. dahliae did not show differential pathogenicity on tolerant potato cultivars. The overall results indicated that (i) a gene similar to Ve does not confer disease tolerance to potato; (ii) the phenotypic tolerance response of potato is similar to the resistance response in tomato; and (iii) the root plays an important role in both tomato and potato, but other general defense mechanisms appear to be present in potato as well. (P)

Possible Resistance to Verticillium dahliae Race 2 in Tomato

J. Gold and J. Robb Dept. of Molecular Biology and Genetics, University of Guelph, Guelph, Ontario NIG 2W1, Canada [Telefax: +I-519-837-2075]

Only limited resistance to Verticillium was available in commercial tomatoes until the introduction of the Ve-gene in 1951. This gene, which seems to be inherited in a monogenic and dominant fashion, provides defense against Verticillium albo-atrum and V. dahliae race 1, but not V. dahliae race 2. In cv. Craigella, near-isolines are available which are resistant (Ve+) or susceptible (Ve-) to V. albo-atrum and V. dahliae race 1. Resistance in Ve+ plants apparently results from the rapid coating of xylem vessel walls with suberin-rich materials, effectively sealing pit membranes and blocking fungal escape from spore trapping sites. V. dahliae race 2 suppresses


this defense response and colonizes extensively in Ve+ plants, resulting in disease development. Cultivar IRAT has been reported variously to possess some resistance against V. dahliae race 2. Light microscope and histochemical studies of IRAT in our laboratory indicate that V. dahliae, races 1 and 2, is able to suppress the vascular coating response as in a susceptible reaction; however, escape of the pathogen from spore trapping sites is severely limited by a marked reduction in sporulation and spore germination capacity of the fungus. This severely curtails the pathogen's ability to colonize both laterally and vertically in the plant and within 4 days secondary colonization is considerably less than is observed in a susceptible interaction. The evidence suggests that cv. IRAT possesses an alternative mechanism of resistance which is effective, at least to some degree, against V. dahliae, races 1 and 2. (L)

Progress in the Search for Verticillium Wilt-Resistant Artichoke

F. Ciccarese, M. Cirulli and M. Amenduni Dept. of Plant Pathology, University of Bari, 70126 Bari, Italy

[Telefax: +39-80-554-2926]

Since the early 1980s, severe outbreaks of wilt caused by Verticillium dahliae have been observed frequently in artichoke-growing areas in southern Italy. Diseased plants exhibit stunting, yellowing, wilting, drying of leaves and vascular discoloration; they do not yield at all or produce only a few buds which are deformed and small. The strategy for controlling Verticillium wilt of artichoke is based on planting in non-infested soil, use of disease-free vegetatively produced plants, and long-term rotation with non-host species. Soil fumigation with methyl bromide or dazomet provides nearly complete control of disease, but this practice is limited by its cost and by ecological constraints. The use of resistant cultivars would be more economic and ecologically sustainable.

A preliminary screening test of 130 artichoke clones originating from several different countries showed a wide range of reaction to Verticillium wilt. Eleven artichoke clones selected in that screening, together with three susceptible clones, were tested in the field over 3 years of perennial cropping to evaluate their resistance/susceptibility to V. dahliae. There were no artichoke clones which showed complete resistance; nevertheless, clones possessing different levels of resistance exhibited a percentage of diseased plants and disease severity indices much lower than those of susceptible clones. Based on external symptoms, artichokes could be classified as resistant (percentage of diseased plants and severity indices below 30% and 20%, respectively), moder~tely resistant (percentage of diseased plants ranging from 30% to 80% and severity indices ranging from 20% to 60%), or susceptible (percentage of diseased plants and severity indices over 80% and 60%, respectively). In general~ the correlation between vascular discoloration and external symptoms was high; however, some resistant clones showed a disproportionally higher percentage of plants with vascular discoloration than did plants with external symptoms, indicating that resistant clones may contain different forms of resistance. The time-course of development of Verticillium wilt during the third year of cropping proved to be crucial in detecting differences between resistant and moderately resistant artichokes. (L)

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A Comparative Study of the Resistance Mechanisms of Cotton ( Gossypium hirsutum ) to Fusarium oxysporum

f.sp. vasinfectum and Verticillium dahliae

S. Eldon and R.J. Hillocks Dept. of Agriculture, University of Reading,

Reading RG6 2AT, UK [Telefax: +44-734-352421]

Vascular wilt diseases of cotton are caused by both Fusarium oxysporum f.sp. vasinfectum and Verticillium dahliae. The foliar symptoms and progress of the diseases are similar. Verticillium is prevalent in cooler climates and has an optimum mean temperature for expression of 23~ Fusarium is more damaging in hotter areas, with an optimum mean temperature for expression of 30~ There is some geographical overlap.

Analysis of inoculated stele tissue showed that hemigossypol (HG) was the most abundantly produced sesquiterpenoid phytoalexin, regardless of cultivar. In vitro experiments showed that HG did not affect the mycelial growth rate of the pathogens. However, even relatively low levels of the phytoalexin (5 ktg m1-1) inhibited germination of conidia from both fungi. This inhibition was shown to be much greater for Verticillium than for Fusarium. The optimum temperature for the germination of conidia was found to be 3.5~ higher for Fusarium than for Verticillium. Higher temperatures were also shown to inhibit rapidly Verticillium germination, whereas Fusarium conidia had a much greater tolerance. These results explain at least partly the difference in the optimum temperatures for disease expression in the field. They also indicate that the rate of sesquiterpenoid phytoalexin accumulation is more important as a resistance factor against Verticillium than against Fusarium. (P)

Field Assessment of Wilt Resistance in Hop

D.A. Chambers 1 and P. Darby 2 1Horticulture Research International, East Mailing, West Malling,

Kent ME19 6BJ [Telefax: +44-732-849067]; and 2Horticulture Research International Hop Research Unit, Wye College, Wye, Ashford, Kent TN25 5AH, UK

Wilt caused by Verticillium albo-atrum threatened to destroy the British hop industry in the 1940s. Since that time resistance to the disease has been a primary selection criterion in hop breeding in the U.K. In spite of the introduction of cultivars of higher resistance to the pathogen, new strains of V. albo-atrum of grater pathogenicity have emerged to threaten hop production anew. The search for genotypes highly resistant to wilt is therefore a continuing goal of the breeding program.

All advanced lines and selections from the breeding program at Wye are assessed for resistance in the field at East Mailing. The inclusion of stem base colonization data in resistance assessment, which was previously based on symptoms alone, has made it possible to amplify the response range of resistant genotypes. This has allowed the selection of several lines which are more resistant than Wye Target, currently the most resistant commercial cultivar. One of these lines, coded TC 105, is being evaluated for commercial potential in farm trials. (P)

Phytoparasifica 23:1, 1995 63

The Mechanisms Causing'Tolerance of Plants to Wilt ~

R.K. Shadmanov Institute of Experimental Plant Biology, Academy of Sciences,

Tashkent, Republic of Uzbekistan

The penetration of a pathogen int6 the plant occurs at the very first period of germination. It must pass, therefore, through the epidermal cell layer of seed covers or rootlets. Due to the existence of active enzyme systems in this layer, intensive oxidative processes occur. The oxidation products formed have a stronger fungitoxic action compared with the initial compounds. Exuding on the surface of the epidermal layer at the points of contact with the infection, they form a powerful chemical barrier. That is why some investigators observed, but were unable to explain, the mass settling by fungi on the root surface, but only a slight penetration into the tissues in plants which are only slightly affected; and, on the other hand, its almost complete absence on the roots of affected plants with a high rate of infection in their tissues.

Depending on the mode of introduction, a number of cells with enhanced enzyme activity of infection either die (immuno genotypes), or get into the vascular system of a plant so weakened or in such small quantities, that they have no essential effect on the processes of normal development (tolerant genotypes). We have found that the mechanism of primary defense from fungus infection is conditioned by two chains of respiration. These are non-mitochondrial chains of respiration, which do not form ATP, and contain as terminal oxidase phenoloxidase and ascorbatoxidase. In covered and epidermal tissues of seeds of tolerant genotypes we have revealed all elements of these respiration systems. In covered seed tissues, affected by the genotype's wilt, they have not been discerned. To date, the function of these two respiration chains in plants is still undefined. (P)

Recovery of Verticillium-Infected Ash Trees

J.A. Hiemstra Dept. of Forestry, Agricultural University, 6700 HB Wageningen, the Netherlands

(Present address: DLO-Centre for Plant Breeding and Reproduction Research [ CPRO-DLO ], 6700 AA Wageningen, the Netherlands)[Telefax: +31-8370-180941

The effect of infection by Verticillium dahliae Kleb. on young ash trees (Fraxinus excelsior L.) was investigated. Disease development of 15-year-old forest trees affected by ash wilt disease (natural infection of V. dahliae) and disease development and growth of inoculated 5-year-old trees were monitored.

Recovery was common among naturally infected ash trees. About half of the diseased trees were without symptoms one year later; this percentage increased in subsequent years. On the other hand, some of the diseased trees showed dieback or died. Recurrence of symptoms in succeeding years was rare. Inoculated trees never died or showed dieback and only one of 102 inoculated trees evinced recurrent symptoms.

Recovery of infected trees is attributed to formation of new uninfected xylem and confinement of V. dahliae to the xylem already present at the time of infection. Dieback and death of infected trees are supposed to be the result of the fungus reaching the cambium and killing it before new uninfected xylem can be formed.


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During the inoculation experiments V. dahliae reduced growth of inoculated trees significantly, the magnitude of the reduction depending on the method and date of inoculation. The effect on diameter growth was greater and longer lasting than on height growth. This has an ecologically important implication for forest stands affected by ash wilt disease. Despite initial recovery, many of the affected ash trees will die because of suppression by healthy neighbors. (L)

F: CHEMICAL, BIOLOGICAL AND OTHER MODES OF CONTROL OF VERTICILLIUM

Methyl Bromide as a Soil Disinfestant for Control of VerticiUium dahliae in Crop Rotat ion

G. Maharshak, 1 L. Klein, 2 D. Sadan, 3 Leah Tsror (Lahkim) 4 and A. Nachmias 4 1Kibbutz Re'im, M.P. Negev, 85132; 2Bromine Compounds Ltd., Be'er Sheva 84101;

3Extension Service, Israel Ministry of Agriculture, Negev Region; and 4Dept. of Plant Pathology, ARO, Gilat Regional Experiment Station,

M.P. Negev 2, 85280, Israel [Telefax: +972-7-926337]

Potato is a major crop in the Negev area of Israel. One of the important limiting factors to potato production in this region is potato early dying (PED) syndrome, caused by the interaction of Verticillium dahliae and the nematode Pratylenchus mediterraneus. Soil disinfestation is a commonly used means to control PED. Methyl bromide (MB) is an effective fumigant that kills fungi, bacteria, nematodes, and seeds of several weeds. However, due to environmental considerations and very high cost, the use of this material is being reviewed. The goal of this study was to reduce the quantities of MB exposed to the environment when applying it during crop rotations.

The field experiments were performed on potato, corn and peanuts in the commercial fields of Kibbutz Re'im in the northern Negev region. There was a significant reduction in disease population in the soil and an increase in commercial yield following fumigation with MB. On the other hand, the damage to the mycorrhizae population was compensated for by an excess amount of phosphoric acid, applied through the water system.

These results encourage us to continue the long-term research on optimization of the use of MB as a soil disinfectant. (P)

Control of Verticillium Wilt with Methyl Bromide at Reduced Dosage Using Virtually Impermeable Films

A. Gamliel, 1 A. Grinstein, 1 L. Klein, 2 I. Peretz 3 and J. Katan 4 l lnst. of Agricultural Engineering, ARO, The Volcani Center, Bet Dagan 50250

[Telefax: +972-3-960-4704]; 2Bromine Compounds Ltd., Agricultural Dept., Be'er Sheva 84101; 3Hevel Maon M.P. HaNegev 85465; and 4Dept. of Plant Pathology and Microbiology, The Hebrew

University of Jerusalem, Faculty of Agriculture, Rehovot 76100, Israel

Soil fumigation with methyl bromide (MB) is a common practice to control Verticillium wilt in many crops. However, fumigation with MB may cause environmental problems; therefore, reducing MB dosage is a desirable goal. The commonly used films for MB fumigation, e.g. low density polyethylene (LDPE), provide a poor barrier, and enable the emission of MB to the


atmosphere during fumigation. The use of virtually impermeable films (VIF) for MB fumigation minimizes this emission during the exposure period and increases the time MB is retained in the soil (T). Using VIF might enable the use of a reduced dosage (C), with an effective CxT product. Co-extruded multilayer films with polyamide or ethylene vinyl alcohol (EVOH) as the barrier layer were found to be virtually impermeable to MB in the laboratory. In microplot-designed field experiments, VIF retained the gas in soil for longer periods. The resultant CxT product originating from reduced dosage of MB under VIF was similar to that of a full dosage of MB applied under LDPE film. Highly effective control of the pathogens Verticillium dahliae, Fusarium oxysporum f. sp. melonis, Sclerotium rolfsii and Pythium spp. was achieved by MB applied under VIF with a reduced dosage of MB (50% or less of the commercially recommended dosage) to a depth of 40 c m .

The effect of VIF with reduced dosage of MB on the control of Verticillium wilt was examined in commercial potato .fields naturally infected with the pathogen. Microsclerotia of V. dahliae were effectively controlled to a depth of 40 cm by fumigation with MB at a rate of 50 g/m 2 using LDPE or 25 g/m 2 using VIF. Control of V. dahliae microsclerotia by MB at a rate of 25 g/m 2 under LDPE was less effective. Verticillium wilt of potato was effectively controlled by fumigation with MB at a rate of 50 g/m 2 under LDPE or a at rate of 25 g/m 2 under VIF, whereas fumigation at 25 g/m 2 under LDPE was less effective. Plants in the nonfumigated plots and in plots fumigated with MB at a rate of 25 g/m 2 and using LDPE, collapsed 4 weeks before harvest. In contrast, plants grown in the plots that were fumigated at full dosage and those fumigated at half rate under VIF, remained viable until harvest. Subsequently, potato yield in the plots fumigated at half dosage with VIF was similar to that obtained in the plots fumigated at full dosage, and was 31% higher than in the nonfumigated plots. The percentage of big tubers (>45 g) was 50-60% in the fumigated plots compared with 30% in the nonfumigated control. Thus, Verticillium wilt also reduces commercial tuber quality. The use of VIF for MB fumigation can thus be effective for soil fumigation while reducing environmental hazards. (L)

Formaldehyde as a Soil Disinfestant for Control of VerticiUium dahliae in Crop Rotation

G. Maharshak, 1 O. Heiman, 2 D. Sadan, 3 S. Varshavski, 4

Leah Tsror (Lahkim) 5 and A. Nachmias 5 1Kibbutz Re'ira, M.P. Negev, 85132; 2Dor Chemicals Ltd., Haifa 26100;

3Extension Service, Israel Ministry of Agriculture, Negev Region; 4Maon District Enterprises, Negev 85465; and 5Dept. of Plant Pathology, ARO,

Gilat Regional Experiment Station, M.P. Negev 2, 85280, Israel [Telefax: +972-7-926337]

Potato is a major crop in the Negev area of Israel. One of the important limiting factors to potato production in this region is potato early dying (PED) syndrome, caused by the interaction of Verticillium dahliae and the nematode Pratylenchus mediterraneus. Soil disinfestation is a commonly used means to control PED. Formaldehyde applied as a soil disinfestant at a concentration of 2500 or 5000 //ha prior to the potato crop, significantly reduces the nematode population in the soil. This treatment also achieves a reduction in Verticillium symptoms and an increase in potato yield. These effects of the soil disinfestation have been observed in the crops that were planted following the first one: either a second crop of potato, or peanuts.

The results encourage us to continue field experiments and to find the optimal use of formaldehyde as a soil disinfestant within crop rotations in the northern Negev area. (P)

66 Meetings

Inhibitory Effects of Volatile Compounds from Rapeseed Meal to Verticillium dahliae

H.A. Melouk, X. Li, J.P. Damicone and K.E. Jackson Dept. of Plant Pathology, Oklahoma State University, and USDA-ARS,

Stillwater, OK 74078-9947, USA [Telefax: +1-405-744-7373]

Decomposition of rapeseed meal (RSM) in moist soil produces volatile compounds with biocidal properties. RSM (containing 36 ~tm/g glucosinolate) was added to moistened soil in glass baking dishes (22x12x6 cm) at rates ranging from 0 to 60 g/kg of soil. Each baking dish was placed in a sealed plexiglass chamber (30x17x10 cm). The chamber was kept at 22~ for 2 days to allow the production of volatile compounds prior to introducing a 9-cm petri dish containing potato- dextrose-agar medium inoculated with a 0.17 cm z plug of Verticillium dahliae. Mycelial growth of V. dahliae was measured after 4, 8, 12 and 18 days of incubation at 22~ Volatile compounds released from RSM at 60 g/kg soil completely inhibited the growth of V. dahliae at all dates of incubation. At 40 g/kg soil, approximately 65% inhibition of mycelial growth was achieved. Formation of microsclerotia by V. dahliae was reduced in cultures exposed to volatile compounds released from RSM at all rates tested. The amount of melanin extracted from V. dahliae cultures exposed to volatiles released from RSM in soil at the lowest rate (20 g/kg soil) was reduced by 99% compared with the control. (L)

Enhancement of Performance of Talaromycesflavus by Combination with Sublethal Metham Sodium or Heat

D.R. Fravel 1 and E.C. Tjamos 2 1Biocontrol of Plant Diseases Laboratory, USDA-ARS, Beltsville, MD

20705, USA [Telefax: +1-301-504-5968]; and 2Dept. of Plant Pathology, Agricultural Unfi~ersity of Athens, Votanikos 118 55, Athens, Greece

Talaromyces flavus has shown promise in the control of Verticillium wilt, although control is inconsistent and often not sufficient to be economically meaningful. This research was undertaken to determine whether control could be enhanced by combining T. flavus with sublethal levels of chemical fumigants or heating. Sublethal treatments may (a) be less disruptive to the soil ecosystem, (b) reduce competitive interactions facilitating establishment of T. flavus, and (c) weaken Verticillium dahliae, making it more susceptible to attack by biocontrol organisms.

Soil containing microsclerotia (MS) of V. dahliae, naked ascospores of T. flavus, or alginate prill with ascospores of T. flavus was drenched with 0, 18 or 90 ~tl metham sodium in 30 ml water per 250 g of soil (approximately equal to 0, 187 or 935 //ha). The low rate of metham sodium reduced the growth rate of colonies resulting from recovered MS but did not affect the viability of V. dahliae. The growth rate of T. flavus from ascospores or prill was not affected by either metham sodium treatment. Populations of T. flavus in prill recovered from either rate of metham sodium did not differ from the untreated control. Naked ascospores of T. flavus treated with the low rate of metham sodium did not lose their viability, whereas those treated with the high rate of metham sodium varied in viability from one experiment to another. Eggplants were drenched at 0, 3 and 5 weeks after seeding with 10 6 ascospores of T. flavus / 7.5-cm-diam pot. At 5 weeks, eggplants were transplanted into soil containing 50 MS of V. dahliae ! g natural field soil treated with metham sodium at the rates stated above. There was a significant interaction between metham sodium and T. flavus, resulting in control similar to the healthy controls and the full rate of the fumigant. In


field tests for control of V. dahliae on eggplant, interactions between T. flavus and metham sodium were apparent on approximately half of the sampling dates.

Microsclerotia in water were not heated, or were heated using one of three regimes to simulate soil solarization. Regimes l, 2 and 3 consisted of 31~ for 10 h followed by 35~ for 14 h; 33~ for 10 h then 36~ for 14 h; or 35~ for 10 h then 38~ for 14 h, respectively. Five days after plating, 93-96% of the MS exposed to 1-5 days of regime 1, respectively, had germinated, whereas 90, 82, 62, 62 and 50% of MS treated for 1-5 days, respectively, to regime 2 had germinated. Heating under regime 3 for 1-5 days resulted in 92, 37, 14, 1 and 0%, respectively, of MS germinated 5 days after plating. Three days after plating, approximately 40% of the colonies from the unheated controls began forming round, melanized MS and 75% had formed MS by 7 days after plating. In contrast, MS heated under regime 3 for 1 day produced colonies which usually formed few melanized MS. Only 5% of these colonies formed melanized MS 6 days after plating and 10% had formed melanized MS 9 days after plating. In greenhouse tests in field soil, there was a highly significant interaction between the effect of heating on the MS and the effect of T. flavus on the MS, reflecting a synergistic interaction between heating and T. flavus. (L)

Distribution and Establishment of Talaromycesflavus, a Biocontrol Agent against VerticiUium dahliae,

in Soil and on Roots of Solanaceous Crops

E.C. Tjamos 1 and D.R. Fravel 2 1Dept. of Plant Pathology, Agricultural University of Athens, Votanikos

118 55, Athens, Greece [Telefax: +30-301-346-0885]; and 2Biocontrol of Plant Diseases Laboratory, USDA-ARS, Beltsville, MD 20705, USA

Talaromycesflavus, a VerticiUium dahliae bioantagonist, may function through a combination of mechanisms, including antibiosis, parasitism and competition. Since the zone of root elongation in plants is both the site of release of glucose and the principal place for root penetration and colonization by germinating V. dahliae microsclerotia, T. flavus establishment in the zone of elongation may result in biocontrol. Previous work indicated that the antagonist preferentially colonizes the rhizosphere of various solanaceous hosts, cotton, artichoke and olive, although the actual location of the antagonist is unknown.

Studies of the spatial distribution of the antagonist in the root zone of tomato, potato and eggplant (Solanaceae) demonstrated that T. flavus is able to colonize the surface of root tips and roots in significantly greater numbers compared with the rhizosphere or with nonrhizosphere soil. Preferential colonization of the root tips was observed for all three solanaceous hosts and for all sampling times. Differences between root tip colonization expressed on a per-gram fresh weight basis, compared with colonization of roots, rhizosphere and nonrhizosphere soil became less pronounced at later sarnpling dates. However, when colonization was expressed as surface area, the difference between root tips and roots was approximately tenfold greater. Vigorous washing of the roots resulted in a significant decrease in colonized root tips, indicating a loose affinity of T. flavus to the root tips. T. flavus applied via root drenching with an ascospore suspension was far more effective in colonizing root tips or roots compared with T. flavus prills. This is the first evidence of preferential establishment of T. flavus in the sites of colonization of T. dahliae. (L)

68 Meetings

Talaromycesflavus as a Potential Biocontrol Agent against Verticillium dahliae in Potatoes

M.P.M. Nagtzaam Dept. of Phytopathology, Wageningen Agricultural University, 6700 EE


Talaromyces flavus was evaluated as a biocontrol agent against Verticillium dahliae in a field experiment by amending soil with 43 kg ha -1 of a product prepared by grinding alginate-wheat bran granoles containing ascospores and at the same time treating the seed potatoes with ascospores in talcum powder before planting. Talcum powder and alginate-bran granules contained a mixture of ascospores of three strains of T. flavus. V. dahliae was applied at a density of 50 microsclerotia (MS) g-1 soil in autumn 1992 or in spring 1993, resulting in different inoculum densities at planting date. Fresh and dry weights of tubers, leaves and stems were assessed by sub-sampling plots once in 3 weeks.

In treated plots, T. flavus was recovered from roots of potato plants sampled at 40 and 136 days after planting. The biocontrol agent reduced stem colonization (P<0.05) by V. dahliae in plots that had been infested with V. dahliae in autumn 1992. T. flavus did not reduce colonization of the roots or the visually assessed density of MS on haulms that had received the commercial application of haulm-killing herbicides. However, in plots that had received V. dahliae in the autumn of 1992, tuber weight was significantly higher (P<0.05) in those treated with T. flavus than in those without the antagonist. This effect on crop yield was not observed in plots that had received V. dahliae in spring 1993. The results indicate that control of V. dahliae is a function of the density of the pathogen to that of T. flavus.

It is concluded that T. flavus has at least some potential to control V. dahliae in the field. Its mode of action against V. dahliae is currently being studied. Further research is aimed at increasing its efficacy in the field by optimizing exposure of the MS of the pathogen to the antagonist and by selecting more effective strains of the antagonist. (L)

Antagonistic Properties of Wild-Types and Mutants of Talaromyces flavus against Verticillium dahliae

E.S. Saito, 1 I.S. Melo, 2 J.L. Azevedo 1 and J.L. Faull 3 1ESALQ, University of Sffo Paulo, Piracicaba-SP, Brazil; 2 Centro Nacional

de Pesquisa de Monitoramento e Avaliaq:ffo de Impacto Ambiental (CNPMA/EMBRAPA), 13820 Jaguariuna-SP, Brazil [Telefax: +55-192-672202]; and

3Birkbeck College, Botany Dept., University of London, London WCIE 7HX, UK

Verticillium dahliae is the most important pathogen of eggplant in Brazil. Despite progress in the development of resistant cultivars, there are currently no commercial cultivars with resistance to V. dahliae. In this context, the objective of the work described here was to evaluate the antagonistic potential of wild-types and mutants of Talaromyces flavus against V. dahliae. The T. flavus strain IG, and the mutant T4, showed strong antagonistic activity, causing over 73% inhibition of growth of V. dahliae. In natural soil these strains significantly reduced the Verticillium wilt, by 44%, whereas in autoclaved soil infested with the pathogen the mutant T4 was consistently effective in reducing the disease and promoted plant growth. The antagonism appeared to result from antibiotic production by Talaromyces. A metabolite extracted with ethyl acetate from culture filtrates demonstrated potent inhibitory activity against V. dahliae, Fusarium solani,


Sclerotium rolfsii, Rhizoctonia solani and Sclerotinia sclerotiorum. Differences were also observed among the strains of T. flavus in the production of lytic enzymes. All T. flavus strains, when characterized cytologically, presented one nucleus per conidium or ascospore. The performance of this antagonist when formulated with pectin from citrus pulp or sodium alginate was compared. (P)

Effect of Rye Intercropping-Enhanced Fluorescent Pseudomonads on the Control of Verticillium Wilt in Hops

Ewa Solarska Institute of Soil Science and Plant Cultivation, 21002 Jastkow

K/Lublina, Poland [Telefax: +48-81-10410]

Fluorescent pseudomonads with enhanced antagonism against Verticillium albo-atrum were isolated from the rhizosphere of hops intercropped with rye and tested in a pot experiment with soil infested with the pathogen. The isolates were found to increase plant weight and reduce the number of diseased plants. Rye intercropped with hops had the same effect on hop plants' weight and disease incidence. (L)

Prospects for Managing Irrigation Water to Suppress Potato Early Dying

M.L. Powelson, 1 M.R. Cappaert 1 and N.W. Christensen 2 1Dept. of Botany and Plant Pathology; and 2Dept. of Crop and Soil Science,

Oregon State University, Corvallis, OR 97331, USA [Telefax: +1-503-737-3573]

In irrigated potato systems, a factor that influences the development of potato early dying (PED) is the amount of water applied. Results of earlier studies conducted at different geographical locations in the United States have shown that PED can be severe if soil moisture content is excessive throughout the development of the crop. Field studies were conducted in the Columbia Basin of the Pacific Northwest in 1991 and 1992 to determine whether the amount of water applied between emergence and tuber initiation affects the severity of PED and tuber yield. The amount of water applied to the moderate irrigation treatment approximated the estimated consumptive use (ECU) of the potato plant. The deficit and excessive irrigation treatments were approximately 50% and 150% of ECU, respectively. During the treatment period, a total of 9.7, 18.1 and 27 cm of water was applied in 1991, and 13.6, 18.1 and 34.9 cm in 1992. After tuber initiation, all plots were irrigated at 100% ECU.

Severity of PED was significantly lower in the deficit compared with the excessive irrigation treatment. The area under the senescence progress curve values was 29.5% and 9.1% lower in the deficit than in the excessive early season treatments in 1991 and 1992, respectively. Tuber yield was 17% higher in the deficit than in the excessive early season treatments in 1991. Irrigation regime had no significant effect on tuber yield in 1992. In the excessive irrigation treatment at 1000 degree-days after planting, disease was 35% more severe in 1991 than in 1992, which may explain, in part, the yield differences between treatments in 1991 but not in 1992. In 1992, 6.2 cm of rain fell during the treatment period, which may have negated the effect of irrigation treatment on tuber yield. Conditions of lower soil water content (ca 70% available soil moisture) between emergence and tuber initiation reduced the rate of plant senescence. It is during this 4-week period that soil moisture can be safely manipulated without jeopardizing tuber quality. (L)

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Soil Suppressiveness of Verticillium dahliae Infection on Potato with Cropping Practices

O.C. Huisman, 1 J.R. Davis, 2 L.H. Sorensen 2 and A.T. Schneider 2 1Dept. of Plant Pathology, University of California, Berkeley, CA 94720; and

2University of Idaho, Research and Extension Center, Aberdeen, ID 83210, USA [Telefax: +1-208-397-43111

Studies conducted in Idaho during the years 1990-1993 showed that the use of cover crops may enable management and control of Verticillium wilt of Russet Burbank potatoes without pesticides. Results indicate disease control, increased yields, improvement in potato quality and size, and increases in soil fertility, with carry-over benefits lasting for up to two years. Ecological studies conducted concomitantly with these investigations implicate biological control, according to the following lines of evidence: the direct effects of treatments upon soilborne VerticiUium dahliae populations do not alone account for disease suppression; and root colonization data indicate V. dahliae suppression on roots to be negatively correlated (P=0.01) with disease incidence and positively with potato yield. Disease reduction was shown to be highly correlated with both nonspecific microbial activities determined by the fluorescein diacetate procedure and by major changes in microbial populations as evidenced by increases of non-pathogenic Fusarium spp. (e.g. F. equiseti and F. oxysporum). To date, our most effective cropping practices have involved green manure treatments of either Sorghum vulgare var. sudanense (Sudan grass) or Zea mays (corn), but benefits have also been shown with Avena sativa (oats) and Brassica napus var. napus (rape).

Following three consecutive years of green manure treatments, soil suppressiveness was found to occur during two consecutive years of potato cropping. In contrast, green manure treatments that had been cropped continuously for only two consecutive years, failed to suppress wilt during the second continuous year of potato. 1993 studies continued to show suppression of Verticillium wilt following 2 years of successive cropping treatments. When a green manure crop of Sudan grass was introduced into the soil during 1991 and followed by a barley crop during 1992, the effect on Verticillium wilt suppression was equivalent to two successive years of Sudan grass green manures. Although soilborne V. dahliae densities were not reduced with treatments, V. dahliae root infections and the incidence of Verticillium wilt were still reduced. Results continued to show a negative correlation between wilt and microbial activity. (L)

H: R&D SCHEMES

Cotton Research & Development Corporation-Funded Research into Verticillium Wilt of Cotton in Australia

S.J. Allen Australian Cotton Research Institute, Narrabri,

New South Wales, 2390 Australia [Telefax: +61-67-931186]

The Australian Cotton Research and Development Corporation (CRDC) is funded jointly by cotton growers and the Commonwealth Government. Growers contribute Australian $1.75 per bale as a research levy, an amount matched by the Government and used to assist in a wide range of research projects. The CRDC has annual funding of approximately $6 million and accounts for more than 40% of all cotton research in Australia.


Verticillium wilt (caused by VerticiUium dahliae Kleb.) is considered to be the most important disease of cotton in Australia. The'CRDC is currently funding seven research projects which either directly or indirectly include aspects of Verticillium wilt. These projects are as follows: (i) Breeding improved cotton varieties - Dr. N.J. Thomson, Dr. G.A. Constable and Mr. P.E.

Reid - Commonwealth Scientific and Industrial Research Organisation (CSIRO), Division of Plant Industry, Australian Cotton Research Institute, Narrabri (ACRI-N).

(ii) Diseases of cotton - Dr. S.J. Allen - NSW Agriculture (ACRI-N). (iii) Survey of cotton diseases in Queensland - Dr. J.K. Kochman - Queensland Department of

Primary Industries, Toowoomba. (iv) Biological control of Verticillium wilt and seedling diseases of cotton - Dr. S.J. Allen and

Dr. V.S. Putcha - NSW Agriculture (ACRI-N). (v) Verticillium wilt of cotton: Identification of fungal pathogens and isolation of potential plant

resistance genes - Dr. B. Lyon - University of Sydney. (vi) Genetic manipulation of natural plant defenses for the biocontrol of Verticillium wilt diseases

of cotton - Dr. E. Dennis and Dr. D. Llewellyn - CSIRO, Division of Plant Industry, Canberra.

(vii) Induced resistance to cotton diseases as part of integrated pest and disease management - Prof. B.J. Deverall - University of Sydney.

In addition to the above, a project entitled "Using glucose oxidase to control Verticillium wilt bf cotton" is being funded by Cotton Seed Distributors Ltd., which a grower-controlled company producing planting seed for the Australian cotton industry.

Information on the relevant objectives of each of these projects was presented. (P)

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M E E T I N G S

ABSTRACTS OF PAPERS PRESENTED AT

THE 7TH CONFERENCE OF THE E N T O M O L O G I C A L SOCIETY OF ISRAEL

Microbial Control of Insect Pests - Research and Application

December 12, 1994 ARO, The Volcani Center; Bet Dagan, Israel

Numerical rearrangement of the Meetings (short, consisting of a few lectures only) Conferences (longer, with an extensive number of lectures), on Agricultural Entomology. The following meetings and conferences have taken place so far:

and

New Date numerical order i March 3, 1981 ii February 7-8, 1983 iii April 5, 1984 iv March 3-4, 1986 v April 9, 1987

vi October 15, 1991

Notation Venue Abstracts published in Phytoparasitica

[

1st Meeting Rehovot 9(3):217-222 (1981) 1st Conference Bet Dagan 11(2): 177-132 (1983) 2nd Meeting Bet Dagan not published 2nd Conference Bet Dagan 14(2):155-174 (1986) Erroneously called Bet Dagan 15(2):149-153 (1987) 3rd Conference (should have been called: 3rd Meeting) 3rd Conference Bet Dagan 20(1):67-85 (1992).

In accordance with this rearrangement, the present conference will be called the 7th Conference of the Entomological Society of Israel (see the Roman numerals in the first column of the Table, above).


Introductory Remarks

Microbial Control of Pests in Israel: Research and Practice

M. Wysoki Dept. of Entomology, ARO, The Volcani Center,

Bet Dagan 50250, Israel [Telefax: +972-3-960-4180]

The Seventh Conference on Agricultural Entomology of the Entomological Society of Israel is dedicated to research and practical implementation of microbiological control agents for pests in Israel. It features contributions covering a broad range of topics, dealing with scientific investigations and practical uses of such agents against vectors of human and animal diseases and against agricultural pests. Topics range from the mode of action, efficacy, engineering and uses of Bacillus thuringiensis (Bt), B.t. israelensis (Bti), to baculovimses, and entomopathogenic fungi and nematodes. Also, the discovery of new species, which are both highly effective against target organisms and environmentally safe is described. Bti is applied presently in Africa, North and South America (especially Canada and the USA), Asia (China) and Europe (Germany). In Africa Bti is employed for the control of black flies (Simuliidae), vectors of onchocerciasis.

The papers which are presented include the factors of mosquito larvicidal activity (plasmids), utilization of ~5-endotoxin, which kills mosquitoes and black flies specifically and efficiently, by encapsulation and gradual release of toxins; bioencapsulation in nontarget organisms such as protozoa ingested by mosquito larvae, the target organism; mode of action (~-endotoxin forms pores in the midgut of susceptible species); production of crystalline inclusions containing several polypeptides that are larvicidal and have a cytotoxic effect on cells; as well as factors affecting the decrease in sensitivity to k-toxins. The Bt var. kurstaki products are widely employed in Israel in practical application against lepidopterous pests in tomatoes, peppers, alfalfa, grapes, dates, apples, pears and avocado. In avocado, Bt products are integrated within a comprehensive biological program against lepidopterous pests and are nontoxic for the beneficial insects released in those orchards. Bt products are used also in pine tbrests, especially in the vicinity of human settlements and close to urban areas.

The Nuclear Polyhedrosis Viruses (NPVs) are potential viral biological control agents against lepidopterous pests. Research on them is directed to the genomic structure, identification of major genes, and genetical engineering to improve their effectiveness. The research on entomopathogenic fungi is concentrated on finding new species and strains for testing against target pests, and the search for more virulent biocontrol agents for utilization in the field. A further topic is the seasonal activity of the entomopathogenic fungi in Israel, which varies with the season, viz., (i) active throughout the whole year; (ii) active during most of the year with the exception of summer; and (iii) active during winter and spring.

The insect-parasitic nematodes are already well known as biological control agents and are produced commercially in several countries. Moreover, the U.S. Environmental Protection Agency has exempted the steinemematids and heterorhabditids from registration and regulation requirements. The research on nematodes centers on advanced release technologies, on new populations less influenced by extreme environmental conditions, on selective breeding, genetic manipulation and gene transfer by microinjection, all with the purpose of improving the effectiveness of these biocontrol agents.

In Israel, which is very concerned about its environment, practical use of biological control agents is highly recommended and is incorporated in every crop in which their efficacy has been demonstrated. It is understood that without basic research the goal of extensive use of microbial pest control agents can not be attained. For this reason, many scientists are working on these areas and have obtained impressive results. (L)

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A: Bti and Bt

Bti for Control of Mosquitoes and Black Flies

J. Margalit and A. Zaritsky Dept. of Life Sciences, Ben-Gurion University of the Negev,

Be' er Sheva 84105, Israel [Telefax: +972-7-2323621

Bacillus thuringiensis (Bt) insecticides are presently used worldwide in both developed and developing countries for the control of agricultural pests and of vectors of human and animal diseases.

Bacillus thuringiensis var. israelensis (Bti) is one strain of the Bt species which is active against mosquitoes and black flies. The emergence and spread of insecticide resistance in many species of vectors, the concern with environmental pollution; and the high cost of the new chemical insecticides have directed increasing attention toward Bti as a very effective biological control agent that possesses the desirable properties of a chemical pesticide: highly toxic to the target organism, able to be mass-produced on an industrial scale, a long shelf life, and transportable.

The crylV class of crystal protein genes, which is active against mosquito and black fly larvae, was isolated from Bti. This class contains four genes (crylVA, B, C and D) that encode polypeptides with predicted molecular masses of 134, 128, 78 and 72 kDa. The crystals are stable in neutral pH and dissolve upon ingestion in the larval midgut, where a pH higher than 9 prevails. The composing polypeptides are activated by specific proteases and act coordinately and synergistically to disrupt the epithelial cells of the larval gut. The larvae consequently are paralyzed and die within a short time, depending upon the amount of toxin ingested.

The environmental safety of Bti has been confirmed in numerous tests. At present, Bti is employed on all continents; nearly one million liters of Bti are applied annually against the black flies - vectors of onchocerciasis. In Europe along the Upper Rhine Valley over 100 communities, inhabited by over 2.5 million people, are protected through mosquito abatement programs utilizing Bti. In the United States and Canada, Bti is being used in numerous abatement districts as well. (L)

Improving Efficacy of the Bacterium Bacillus thuringiensis var. israelensis as a Mosquito Control Agent

1 1 2 Z. Barak, A. Zaritsky, A. Markus, S. Boussiba, S. Cohen, 1 2 1 1 M. Grinberg, R. Manasherob and E. Ben-Dov

1Dept. of Life Sciences and 2The Institute for Applied Research. Ben-Gurion University of the Negev,

Be 'er Sheva 84105, Israel [Telefax: +972-7-2762011

The bacterium Bacillus thuringiensis var. israelensis (Bti) produces a ~5-endotoxin that specifically and efficiently kills mosquito and black fly larvae, and is thus utilized for their biological control. Bti's major disadvantage as a control agent is its short persistence under field conditions due to fast sinking into and adsorption onto mud and inactivation of the toxins by solar near-UV light. We attempted to prolong the persistence of Bti as a mosquitocidal agent in nature using three different approaches:

*L = lecture sessions; P --- poster (market place) sessions.


(i) Encapsulation of Bti and its ~-endotoxin in formulations with UV protectants from which the toxins are gradually released into the larval ecosystems.

(ii) Bioencapsu|ation of intact Bti toxins in protozoans that concentrate and actively deliver them to the target organisms, mosquito larvae.

(iii) Expression of Bti fi-endotoxin's genes in microorganisms that inhabit natural waters and are digested by mosquito larvae.

Of the various polymers tested for encapsulation of Bti, polyethylene yielded the best results. A novel formulation was developed from this polymer that released Bti and continued to kill mosquito larvae for over a month in muddy waters exposed to sunlight.

The ciliate protozoan Tetrahymena pyriformis (Tp) concentrated ~-endotoxin in its food vacuoles. Mosquito larvae fed on Bti-loaded Tp (100-300 Bti spores and crystals per cell) died sooner and at lower concentrations than with Bti alone. This enhancement of larval mortality was pronounced at low Bti concentrations, with late-fourth-instar larvae, and with surface-feeding species such as Anopheles stephensi.

Three of Bti's toxin genes were cloned in all seven possible combinations for expression in Escherichia coli; larvicidal activities of the combinations crylVA+crylVD were the highest.

Our investigations are expected to open the door for production of new, efficient formulations of Bti and make its use worthwhile, economically and ecologically. (L)

Raising Activity of Bacillus thuringiensis var. israelensis 5-Endotoxin Against Anopheles stephensi

Larvae by Encapsulation in Tetrahymena pyriformis

R. Manasherob, E. Ben-Dov, J. Margalit, Z. Barak and A. Zaritsky Dept. of Life Sciences, Ben-Gurion University of the Negev,

Be'er Sheva 84105, Israel [Telefax: +972-7-2762011

The bacterium Bacillus thuringiensis vat. israelensis (Bti) is used for specific control of mosquito and black fly larvae, vectors of many infectious diseases. Its larvicidal activity is included in polypeptides of a parasporal crystalline body (~-endotoxin), produced during sporulation. The use of Bti is, however, limited by the low efficacy of current preparations under field conditions, in which it does not reproduce.

To raise its persistence, a commercial powder of Bti was encapsulated in Tetrahymena pyriformis (Tp), a protozoan in which the toxin remains stable. Each Tp cell concentrates in its food vacuoles between 180 and 300 Bti spores and crystals, and keeps them at the water surface. The Tp cells are motile, and thus deliver the toxicity to mosquito larvae, which die upon ingesting them.

Various mosquito species exhibit different levels of susceptibility to Bti. Larvae of most Anopheline species are ten times more tolerant to Bti formulations than are those of Culex and Aedes. Sinkiffg of the toxin and slower feeding rates have been cited as the reasons for the reduced sensitivity. The Tp bioencapsulation system concentrates Bti spores and their toxicity and keeps them at the water surface; it is thus expected to enhance Bti activity against Anopheles larvae.

Bioencapsulation at concentrations of 2.105 spores (2 ~tg) and 500 Tp cells per ml improved the larvicidal activity against Anopheles stephensi: Death set in earlier (lag-time was shorter) and involved much faster 50% and 80% of the exposed populations (LTs0 and LTs0, respectively). The toxicity amplification factor (the ratio between larval death time caused by Tp-encapsulated Bti and that by Bti alone) was 7.4 for LTs0 and 8.7 for LTs0.

Optimum larvicidal activities were achieved with spore/cell ratios of 200-500. The LTso obtained for fourth-instar An. stephensi larvae was 0.43 p.l ml -l with the bioencapsulation system, and 1.66 I.tg ml -I with Bt/alone in a 100-ml test system. Thus, the system decreases fourfold the

76 Meetings

Bti concentration needed to kill 50% of exposed larval populations. Attaining the full advantage of this bioencapsulation system may need larger and deeper test systems.

The increased mortality rate of An. stephensi larvae by encapsulating ~-endotoxin in Tp cells is very likely the consequence of concentrating large quantities of spores and crystals and their efficient delivery to the target organism. Shortening larval mortality time by this bioencapsulation should reduce the exposure time of Bti to detrimental field conditions that inactivate its larvicidal activity, thus raising its efficacy. (P)

Mosquito Larvicidal Activity of Escherichia coli with Combinations of Genes from Bacillus thuringiensis var. israelensis

E. Ben-Dov, S. Boussiba and A. Zaritsky Dept. of Life Sciences, Ben-Gurion University of the Negev,

Be ' er Sheva 84105, Israel [Telefax: +972-7-276201]

Serovar H-14 of Bacillus thuringiensis var. israelensis (Bti) harbors several plasmids. A 75 MDa plasmid is responsible for Bti's mosquito larvicidal activity; it includes the genes crylVA, B, C, and D and cytA, encoding the respective ~-endotoxin proteins (134, 128, 78, 72 and 27 kDa) which are expressed during sporulation, resulting in the paracrystalline toxin. The gene for a regulatory 20 kDa polypeptide, which is required for efficient production of the structural proteins, is mapped on the same plasmid.

The genes for CrylVD and the 20 kDa polypeptide were isolated, separately and together, from a 9.7 kb HindlII fragment, and subcloned in pUC19. A second toxic gene (crylVA) was amplified by means of the Polymerase Chain Reaction and subcloned similarly. All three genes were cloned either alone or in combinations into expression vector pT7 behind the strong T7 promoter and transformed to Escherichia coli strain BL21, which contains on its chromosome the T7 gene 1 coding for T7 RNA polymerase. These genes and their combinations were cloned into expression vector pUHE behind a promoter of an early bacteriophage T7 gene, recognized by E. coli RNA polymerase (strain XL1-Blue MRF').

Expression of crylVA in pUHE24 was verified by bioassays with larvae of Aedes aegypti. Highest mortality (98%) was obtained after 45 min of induction and remained at that level for at least 4 h. Expression of crylVA in pT7 yielded much lower larvicidal activity (80% mortality, but with sixfold higher cell concentrations) after 45 min of induction. At longer induction times, mortality decreased slightly and stabilized at 55% for at least 3.5 h. Expression of gene combinations crylVA and crylVD with or without the 20 kDa protein gene in pUHE24 achieved fourfold higher toxicity than crylVA alone in the same vector.

Five min induction of the combination of crylVD with the 20 kDa protein gene in pT7 gave maximal mortality of approximately 40%, which decreased rapidly and disappeared completely after 50 min, whereas induction of either crylVD or the 20 kDa protein gene alone had no larvicidal activity. CrylVD is thus degraded in E. coli, and partially stabilized by the 20 kDa regulatory protein. Induction in pUHE20 of each of the three clones containing crylVD and the 20 kDa protein gene, together or alone, had no larvicidal activity. Comparison in the same experiment demonstrated that toxicity of crylVA is twofold more toxic than crylVD when expressed from the same vector, and that the corresponding protein (CrylVA) is much more stable than CrylVD in recombinant E. coli. (P)


'Segment Analysis' Approach to Study the Mode of Action of the Bacillus thuringiensis var. israelensis Cytolytic Toxin

E. Gazit, Z. Oren and Y. Shai Dept. of Membrane Research and Biophysics, The Weizmann Institute of

Science, Rehovot 76100, Israel [Telefax: +972-8-3427111

The Gram-positive spore-forming bacterium Bacillus thuringiensis var. israelensis (Bti) produces crystalline inclusions that are highly toxic to larvae of mosquitoes and black flies. These inclusions contain several polypeptides that have been shown to be larvicidal and to have a cytotoxic effect on a wide range of ceils. A 27 kDa toxic protein (Bti toxin) and its corresponding gene have been purified and cloned from Bti isolates. To gain insight into Bti toxin's mode of action, three putative hydrophobic 22-amino-acid peptides were synthesized and characterized spectroscopically and functionally. One segment corresponded to the putative amphiphilic ~- helical region (amino acids 50-71, termed Helix 1), another to amino acids 110-131 (termed Helix 2), and the last to amino acids 149-170 (termed Helix 3). Circular dichroism spectroscopy revealed that whereas Helix 1 and 2 segments adopted high or-helical content in a hydrophobic environment, in agreement with previous models, the putative Helix 3 did not. To monitor peptide-lipid and peptide-peptide interactions, the peptides were labeled selectively with either NBD (7-nitro-2,1,3- benzoxadiazole-4-yl) (an environment-sensitive probe, serving to facilitate binding experiments, and as an energy transfer donor) or tetramethylrhodamine (serving as an acceptor), at their N- terminal amino acids. Both Helix 1 and Helix 2 segments bind strongly to small unilamellar vesicles (SUV), composed of zwitterionic phospholipids, with surface partition coefficients in the order of 10aM -1. The shape of the binding isotherms indicates that Helix 2 forms large aggregates within phospholipid membranes. Resonance energy transfer experiments demonstrated that Helix 1 and Helix 2 segments self-associate and interact with each other, but do not associate with unrelated membrane-bound peptides. Functional characterization demonstrated that Helix 2 permeates phospholipid SUV with a potency similar to that of naturally occurring pore-forming peptides, Helix 1 had lower permeating activity, and Helix 3 did not have any significant permeating activity. Thus, the results support a role for Helices 1 and 2 in the assembly and in the pore formation by Bti cytolytic toxin. (P)

Partial Restriction Mapping of the 72 MDa Plasmid of Bacillus thuringiensis var. israelensis

N. Peleg, G. Nissan, E. Ben-Dov, S. Boussiba and A. Zaritsky Dept. of Life Sciences, Ben-Gurion University of the Negev,

Be 'er Sheva 84105, Israel [Telefax: +972-7-276201]

Bacillus thuringiensis var. israelensis (Bti) is a Gram-positive bacterium which produces parasporal proteinaceous crystals (~-endotoxin) with high specific activity against mosquito larvae. However, Bti's larvicidal efficacy is lost shortly after release in nature. One way to extend persistence of the activity is by expressing cloned crystal's genes in other organisms.

The ~-endotoxin is composed of at least five polypeptides encoded by five genes which are localized on Bti's large (approximately 125 kb) endogenous plasmid. Basic information about control elements and their interactions with the genes is still not satisfactory. A full restriction map of the plasmid will locate all the genes involved and improve our understanding of their interactions.

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Four linkage groups (with sizes of approximately 30, 35, 37 and 48 kb) have already been mapped with SacI and BamHI restriction enzymes. The aim of this project was to complete the circular map of the plasmid by aligning these four groups.

In order to achieve this goal, Psfl and KpnI libraries of the plasmid were constructed, and fragments containing recognition sites of BamHI or Sad were found and isolated. Each was labeled by nick-translation with 32p and hybridized to Southem blots of gels with fragments generated by cleaving the plasmid by several restriction enzymes. The autoradiogram of each probe can align at least two fragments of the relevant enzyme.

The four linkage groups were aligned, thus constructing a full plasmid linear map. More probes are required in order to 'circularise' the map. (P)

Uses of Bacillus thuringiensis for Control of Agricultural Pests in Israel - Present Status and Prospects

A. Navon Dept. of Entomology, ARO, The Volcani Center,

Bet Dagan 50250, Israel [Telefax: +972-3-960-4180]

Within the last 20 years, Bacillus thuringiensis (Bt) has been introduced into pest management programs against moths which are pests of several important agricultural crops. The use of Bt started in the 1960s, when a commercial product of the microbe proved effective against Thaumatopoea wilkinsoni in pine orchards. Since the early 1970s, commercial preparations of Bt based on the subsp, kurstaki have been registered and used effectively against many pest species. In avocado, moths were controlled at commercial levels mostly in Western Galilee and this microbial pest management enabled entomophagous control of Bt-non-target pests, such as scales wiff~ parasitic wasps. With this strategy, the use of chemicals in pest control in avocado was avoided. Bt became useful also in the control of moth pests of bioorganically and conventionally grown vegetables, mostly of cruciferous and solanaceous varieties. The microbe was active in fruit crops, mainly against Cryptoblabes gnidiella and Lobesia botrana. Potency bioassays based on recording the activities of the spore-crystal complex were used to select Bt strains against Helicoverpa armigera, Spodoptera littoralis, Agrotis ipsilon, Boarmia selenaria, C. gnidiella and L. botrana. Rational use of Bt was improved with increasing knowledge in the following areas: (i) Effect of larval feeding behavior on the efficiency of pest control; (ii) differences in susceptibility of larval instars to Bt; (iii) specificity and dose-response of moth species; and (iv) persistence of Bt under different environmental conditions and on different plants.

An increase in the use of Bt in microbial control is expected in view of the global effort to replace chemicals by biological means and the higher production of bioorganic products for export. This will lead to rational uses of new Bt products based on genetic manipulations and new formulations of the microbe. (L)


Development of Granular Bacillus thuringiensis Larval-Feeding Bait Formulations

A. Navon 1 and A. Grinstein 2 1Dept. of Entomology and 2Laboratory for Research on Pest Management

Application, ARO, The Volcani Center, Bet Dagan 50250, Israel [Telefax: +972-3-960-4180]

A new Bacillus thuringiensis product, 'Bt Plus', was developed for moth control in field and vegetable crops. Small-scale experiments showed that the Bt larval-feeding bait formulations controlled larval pests effectively with use of net microbe concentrations lower than the doses recommended for spray applications. Potency bioassays were used to select Bt strains of subsp. kurstaki - with high activity against Helicoverpa armigera and Agrotis ipsilon, and of the subsp. aizawai - active against Spodoptera littoralis. Also the commercial product 'Dipel' (strain HD-1) wettable powder, with 16,000 IU/mg, was used as the microbe source in the formulation. The feeding bait was based on an oily feeding stimulant for the larvae which was selected by means of a choice feeding test with the stimulants offered on polystyrene lamellae. The residual recovery of granules on the plant after application was determined by marking them with a yellow fluorescent powder, dispersing them in an exposure chamber tower and counting the granules under magnification with an ultraviolet lamp. Granules sized 100-150 Ix adhered better to the plant after shaking than did those of greater size. Prewetting of the leaves increased the count of larger granules on the leaves. The Bt formulations were produced by Yewnin-Yoffe Industry Ltd., Israel, in two forms: powders for dusting, and granules, sized 6' 104 and 1.105/kg. Leaf-cage experiments in the field showed that the dust formulation was active in cotton against both H. armigera and S. littoralis" it was effective also against moth pests in tomato. The granular formulation dispersed on the soil was effective against mature larvae of A. ipsilon in bioorganically grown cabbage and has the potential to manage other soil-inhabiting moth larvae. (P)

Factors Affecting Decrease in Sensitivity of Spodoptera littoralis Larvae to Bacillus thuringiensis fi-Endotoxins

2 1 1 M. Keller, 1 N. Strizhov, Avital Regev, B. Sneh, C. Koncz, 2 J. Schell 2 and Aviah Zilberstein 1

1Dept. of Botany, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv 69978, Israel [Telefax: +972-3-640-9380];

2 and Max-Planck-Institut fiir Ziichtungsforschung, Ki~ln, Germany

Denaturation and renaturation (D/R) of ~5-endotoxin crystal proteins produced by Bacillus thuringiensis reduced their capability to kill larvae of Spodoptera littoralis, but larval weight was reduced with increasing toxin concentrations. CryIC produced by an Escherichia coli clone behaved like a D/R crystal in this respect. Quantitative and qualitative differences in degradation of ~-endotoxin by larval midgut proteases between young and advanced larval instars, may provide an explanation for the difference in their susceptibility to the fi-endotoxin. Using specific protease inhibitors such as PMSF (phenylmethylsulfonyl fluoride) and leupeptine indicated that gut juice of 5th instars contained relatively more serine proteases than that of 2nd instars, which may explain the loss of sensitivity of the 5th instars to the CryIC toxins.

The larval population of S. littoralis responded differently to ingestion of CryIC and CryIE. While all individuals in the larval population were equally sensitive to CryIC, one group of the larvae was sensitive to CryIE similarly to that of the whole population to CryIC, whereas the other

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group was insensitive to CrylE. The two groups were reared separately. Brush border membrane vesicles (BBMVs) prepared from midgut epithelia of the general population as well as from the two different populations were used for binding experiments with fluorescein-labeled CrylC and CrylE, for the identification of the two distinctly different receptors. The binding was monitored by reading the fluorescence of washed BBMVs reacting with the labeled toxins. Binding affinity of CrylC to BBMVs made from the general larval population was higher than that of CrylE. However, the affinity of CrylE was much stronger to the BBMVs prepared from sensitive larvae than those from the insensitive ones, compared with the affinity to that of CrylC. It is therefore concluded that there is a direct correlation between binding affinity and insecticidal activity of CrylE, indicating the presence of two distinctly different receptors on the midgut epithelium membrane for CrylC and CrylE. Further work is required to isolate and characterize the two different receptors. (L)

Molecular Mechanism of Membrane Destruction by Bacillus thuringiensis 8-Endotoxin

Y. Shai and E. Gazit Dept. of Membrane Research and Biophysics, The Weizmann Institute of

Science, Rehovot 76100, Israel [Telefax: +972-8-3427111

The 8-endotoxin proteins are produced by Bacillus thuringiensis (BO bacteria during sporulation, and have been widely used as biological insecticides for over two decades. In spite of the vast amount of information on the function of the toxins, their mode of action is still unclear, and is the subject of various studies. The toxins are released as protoxins, solubilized in the midgut and activated by gut proteases. The high specificity of the various ~-endotoxins is assumed to be due primarily to the high affinity of the toxins to specific receptors in the midgut epithelium of susceptible insect species. It is hypothesized that 6-endotoxins form pores, in the midgut epithelium of insects of susceptible species, that eventually cause the insects' death. This mechanism is supported by the finding that activated ~-endotoxins form single ion channels in planar lipid bilayers. This mode of action is also supported by the structure of the CrylIIA 8-endotoxin, determined by X-ray crystallography, that reveals three domains. One of them (domain I) is composed of a bundle of six o~-helices surrounding a central helix (termed Ix-5), and is believed to be the pore domain. We have utilized a synthetic peptide approach combined with fluorescent studies to investigate the mechanism by which 8-endotoxin makes pores in phospholipid vesicles. Peptides corresponding to the highly conserved ct-5 and c~-7 were synthesized and structurally and functionally characterized. The results are consistent with an 'umbrella' model suggested by Ellar and co-workers, for the pore-forming activity of ~i-endotoxin, where ct-5 has transmembrane localization and may be part of the pore lining segment(s), while or-7 may serve as a binding sensor that initiates the binding of the pore domain to the membrane. (L)

Phytoparasitica 23:1~ 1995 81

B: VIRUSES

Baculoviruses Pathogenic to Lepidoptera: Biology and Applicative Projections

N. Chejanovsky, 1 N. Zilberberg, 2 E. Zlotkin, 3 H. Rivkin 1 and M. Gurevitz 2

1Dept. of Entomology, ARO, The Volcani Center, Bet Dagan 50250 [Telefax: +972-3-960-4180]; 2Dept. of Botany, Tel-Aviv University,

Ramat Aviv 69978; and 3Inst. of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

Baculoviruses are pathogenic to insects and do not infect mammals. Their limited host range allowed their registration as bioinsecticides in many countries, since the viruses did not pose any danger to the environment. Major limitations to the utilization of baculoviruses as bioinsecticides are (i) their slow speed of action to kill the targeted insect when compared with chemical insecticides; and (ii) their narrow range of host specificity. An improvement in the baculoviruses' insecticidal activity can be obtained by constructing recombinant viruses carrying 'foreign genes', the expression of which in the infected larvae will eventually result in blocking of vital activities, viz., feeding, movement and breathing. An example of such foreign genes is the anti-insect toxin genes derived from scorpion venom. We have isolated cDNAs coding for the main types of anti- insect toxins from mRNA extracted from the venom gland of the yellow scorpion Leiurus quinquestriatus hebraeus. A baculovirus, the Autographa californica Multiple Nuclear Polyhedrosis Virus (AcMNPV), was engineered to carry the gene coding for the anti-insect alpha toxin LqhoclT. Bioassays performed in our laboratory show that this recombinant virus kills 50% of a Heliothis armigera larval population in approximately 40% of the time required by the wild- type virus to perform the same task. The efficacy of the recombinant virus can be enhanced further by genetic manipulation of the virus and the toxin gene.

A parallel study is being conducted by us in order to overcome the second limitation of baculoviruses (narrow range of host specificity). We are trying by genetic manipulation of the viral genome, to construct baculoviruses which possess a wider range of insect hosts. (L)

Genomic Organization of the Spodoptera littoralis Nuclear Polyhedrosis Virus

O. Faktor Dept. of Entomology, The Hebrew University of Jerusalem, Faculty of

Agriculture, Rehovot 76100, Israel [Telefax: +972-8-4667681

The Spodoptera spp. are major cosmopolitan pests. The identification of nuclear polyhedrosis viruses (NPVs) specific for these pests harbors a potential use for their control. The Spodoptera littoralis multiple NPV (SIMNPV) has an advantage over other Spodoptera NPVs by its ability to infect several Spodoptera spp. However, as with most NPVs, the action of S1MNPV is slow, relative to chemical pesticides. Genetic engineering is the suggested methodology to enhance viral activity. The basic requirement for the design of a genetically improved virus is knowledge of its genomic structure and identification of the major genes. The available information on NPVs' genome organization and genes suitable for manipulation derives from study of the Autographa californica multiple NPV (AcMNPV). This virus contains approximately 150 open reading frames. Of these, structural genes such as the polyhedrin, plO and a gene coding for the ecdysteroid UDP-

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glucosyltransferase (egt) were found in the genome of other NPVs. Since NPVs were subjected to structural changes in their genome through evolution, the localization of these genes in each virus can serve to characterize its genome. In order to study the S1MNPV genome, we used AcMNPV DNA probes for the polyhedrin and egt genes and identified the corresponding genes in S1MNPV. We also localized the plO and p74 genes on the viral genome. Comparison between S1MNPV and other NPV genomes reveals an unusual genome organization. In addition, the sequence of the DNA fragment containing the polyhedrin gene was found to have unique features in and around the coding region. The identification of these genes forms the basis for a genetic approach for improving viral pathogenicity. (L)

C: FUNGI

Seasonal Fluctuations in the Activity and Ecology of Entomopathogenic Fungi Attacking Arthropods in Israel

I.S. Ben-Ze 'ev Plant Protection and Inspection Services, Ministry of

Agriculture, Bet Dagan 50250, Israel [Telefax: +972-3-968-1507]

The life-cycle of a generalized entomopathogenic fungus (EF) can be imagined as a circle composed of two equal or unequal arcs, one describing the EF's 'active season', the other describing the 'rest (dormant) season'. The active season is a chain of shorter cycles, each being completed within several days, and initiating the following one. The continuity of this chain depends upon the availability of hosts and on a suitable climate/microclimate. Each short cycle has two stages: (a) a dispersal stage during which the EF produces and disperses its infective propagules (conidia, sporangiospores, resting spores) and infects the host; and (b) a vegetative stage, during which the EF digests the tissues of the host, increasing its biomass until the host dies. It then produces its dispersal mechanism: conidiophores or sporangiospores and/or other organs with roles in dispersal.

The rest season is that time of the year during which hosts are not available and/or the climate/microclimate is unsuitable for the EF's dispersal or infection of hosts. With the deterioration of one or more of these necessary environmental conditions, the EF enters the rest (dormant) stage. The trigger for this can be the age of or physiological changes in the host, changes of the photoperiod, temperature or humidity,~The fungus reacts to these triggers by a sudden or gradual transition from the production of dispersal propagules to resting ones: resting spores, sclerotia, or resting mycelium in mummified hosts. The awakening of resting propagules is usually triggered by changes in temperature and/or by increased humidity. Resting propagules germinate to produce germ-conidia, the primary infective propagules that initiate the first short cycle of the active season.

The entomogenous mycoflora of Israel can be arranged, according to seasonal activity, into three main categories: (i) EFs with short seasonal activity, usually active during certain parts of winter and/or spring. This group comprises Erynia nouryi, E. occidentalis, E. phalloides and E. phytonomi. (ii) EFs which are active most of the year, with the exception of summer. Members of this group are Entomophthora planchoniana, active during October-June, and Erynia neoaphidis, which is active during November-May. (iii) EFs which are active throughout the year, such as Neozygites fresenii and Erynia radicans.

Seasonal fluctuations in the activity of EFs in Israel are discussed with the help of the following examples: Erynia phytonomi on the alfalfa weevil, Hypera variabilis; Neozygitesfresenii


on the aphid Aphis citricola; and the complex of subspecies known as Erynia radicans, on a number of different hosts. The possibility of intentionally advancing the onset of epizootics in pest populations, by simulation of rain and manipulation of the humidity and microclimate in the target crop, are discussed as well. The overall activity of entomopathogenic fungi in Israel is low during the dry and hot summer, and high during the rainy winter and spring months, November-April. (L)

Biological Control Experiments with Fungal Pathogens of the Sweetpotato Whitefly, Bemisia tabaci

Galina Gindin, 1 I. Barash, 2 I.S. Ben-Ze'ev 3 and B. Raccah 1 l lnst. of Plant Protection, ARO, The Volcani Center, Bet Dagan 50250 [ Telefax: +972-3-960-4180]; 2Dept. of Botany, Tel-A viv University, Ramat Aviv 69978; and 3plant Protection and Inspection Services,

Ministry of Agriculture, Bet Dagan 50250, Israel

Among several fungal pathogens known to attack the sweetpotato whitefly, Bemisia tabaci (Gennadius), the most intensively studied is Verticillium lecanii. Forty strains of V. lecanii isolated in Israel and abroad from different insects were tested for their pathogenicity to eggs, larvae and adults of B. tabaci. Eggs were found to be practically immune, whereas 36% and 53% of larvae and adults, respectively, died of mycosis. Several strains of the fungus, which attacked the host quicker than others (with an LTs0 of 3.2-3.5 days), were selected for further study~

Of a number of other entomopathogenic Deuteromycetes tested on B. tabaci larvae, strains of Beauveria bassiana, B. tenella, Paecilomyces sp. and P. farinosus showed high pathogenicity (LTso = 3.5-5.0 days), while Metarhizium anisopliae was not pathogenic.

Four of the fungal pathogens isolated by us from B. tabaci in glasshouse environments in Israel during 1990-94, had not been reported before from this host. Conidiobolus sp. and C. coronatus (Zygomycotina: Entomophthorales) were isolated from adults. Artificial inoculation experiments with these two species revealed that eggs of B. tabaci were immune to infection and that adults were much more susceptible than larvae. The maximum mortality of adults was ca 30% caused by Conidiobolus sp., and 95% by C. coronatus.

The other two new pathogens are Deuteromycetes, only tentatively identified as 'Hirsutella' sp. and 'Verticillium' sp. The first fungus was isolated several times from both B. tabaci and the aphid Myzus persicae, in mixed populations. This pathogen ldlled B. tabaci larvae and adults in 4 days, causing 44% and 66% mortality, respectively, but did not infect eggs. 'Verticillium' sp. was isolated in 1991 from an epizootic observed in a laboratory rearing ofB. tabaci. This fungus has an LTs0 of 3.5-4.0 days and an epizootic potential higher than that of V. lecanii. In contrast .with the other fungi tested in this study, 'Verticillium' sp. attacks all stages of B. tabaci, eggs included, and grows equally well at 25~ and 30~

The prospects of utilizing entomopathogenic fungi for biocontrol of B. tabaci are discussed. (L)

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D: NEMATODES

Insect-Parasitic Nematodes: Research and Applications

I. Glazer Dept. of Nematology, ARO, The Volcani Center, Bet Dagan 50250, Israel

[Telefax: +972-3-960-41801

Entomopathogenic nematodes of the families Steinernematidae and Heterorhabditidae are lethal insect parasites and are promising agents for the biological control of insect pests. These parasites are mutualistically associated with bacteria of the family Enterobacteriaceae. Third-stage infective juveniles locate and infect suitable insects in the soil. The nematodes release the bacteria into the hemocel within a few hours after penetration. The bacteria multiply, killing the insect within 24-48 h. These nematodes have already been used successfully on high-value crops. Their application provided a level of control for many important insect pests comparable to that of chemicals. Many qualities render nematodes excellent biocontrol agents: they have a broad host range, possess the ability to search actively for the host, and present no hazard to mammals. Finally, the U.S. Environmental Protection Agency (EPA) has exempted entomopathogenic steinemematids and heterorhabditids from registration and regulation requirements, thus simplifying considerably the application, development and commercialization of these nematodes. They are currently considered the second most efficient biocontrol agent against insect pests, after Bacillus thuringiensis. The technology of mass rearing of these nematodes is already well established.

Entomopathogenic nematodes have suppressed insect pest populations in environmentally favorable agroecosystems. However, unfavorable conditions, such as drought, heat and ultraviolet light, limit the broad application of these biocontrol agents. Extensive research is being carried out to overcome these limitations; advanced spraying technology and new formulations may help to solve the problem. New nematode populations which were isolated from regions characterized by extreme environmental conditions are able to tolerate some of the unfavorable situations. Finally, traditional .selective breeding, genetic manipulation, as well as advanced gene transfer by microinjection may be utilized to enhance the use of these nematodes under adverse conditions. (L)

Study presented by title only Ben-Ze'ev, I.S., Gindin, G., Barash, I. and Raccah, B. Identification of entomopathogenic fungi

attacking Bemisia tabaci in Israel. (P)


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