Host range and implications of plant infection by Agrobacterium rhizogenes

Critical Reviews in Plant Sciences, 10(4):387421 (1991)

John R. Porter, PhD. Department of Biological Sciences, Philadelphia College of Pharmacy & Science, Phitadelphia, PA 191 04-4495

Referee: Hector Flores, Ph.D., Department of Plant Pathology, Biotechnology Institute, Penn State University, University Park, PA 16802

ABSTRACT: Agrobacterium rhizogenes is the bacterial agent of "hairy root" disease of many higher plants. This bacterium has been used for insertion of selectable markers into host plants, the culture of immortal root lines for secondary metabolite production, and studies of root physiology and plant-fungd and plant-nematode interactions.

This review reports the results of infection trials, including 36 in the author's laboratory, of 183 varieties, 463 species, 109 families, 49 orders, 5 classes, and 2 divisions. Contrary to earlier reports, A, rhizogenes is infectious on a high percentage (57%) of those plants tried, and neither primitive nor advanced plants are, as a rule, resistant. Four families have been identified which should be considered resistant from the number of unsuccessful trials on several species inoculated with multiple bacterial strains. These are the Cactaceae, Ges- neriaceae, Lamiaceae, and Liliaceae. However, the latter two families may be revised on further work. The Solanaceae, Rosaceae, Fabaceae, Crassulaceae, Caesalpinaceae, Brassicaceae, Polygonaceae, and Asteraceae are all highly susceptible to infection. No chemical basis could be found to explain susceptibility or resistance.

The strain specificity of the bacterium was also reviewed with an examination of the infection percentages of each of the tested strains. Successful infection descended in the order K47 = K599 = HRI > TR105 > ATCC 15834 > A4 > NCPPB 1855 > NCPPB 2655 > TRIO1 > NCPPB 8196 > 232 > ATCC 11325 > ATCC 43057 > TR7 > TR107 = ATCC 13332 = ATCC 13333 for the naturally occurring strains and A41 ARCx > C58C1 (pRiA4) > C58C1 (pRi15834) > C58C1 (pRiTR105) > BL3 - >> C58C1 (pRi8196) for genetically engineered strains. In general, naturally occurring strains were superior to engineered strains in effecting plant transformation.

KEY WORDS: agrobacteria, agroinfection, bacterial strains, hairy-root, plant biosystematics, plant infection.

f . INTRODUCTION

Agrobacteriurn rhizogenes is a phytopathogenic bacterium which infects a number of dicot species and typically causes the disease known as infectious "hairy-root' '; the disease is so called because of the characteristic diageotropic roots, with numerous root hairs, produced at the infection sites.

The development of roots on the host plant is a result of the transformation of infected cells by DNA from the Ri plasmid, the T-DNA, from one or more bacterial cells. l y 2 Because the callus formation due to infection by A. tumefaciens, a

closely related species, has been shown to be due to the presence of genes coding for cytokinin synthesis on the Ti p l a~mid ,~ it has been pre- sumed that a similar synthesis, in this case presumably an auxin, was the cause of the rooting characteristic of ' ' hairy-root ". In refutation of this, Shen et alq4 have shown that transformed roots actually contain less auxin than untrans- formed roots and the transformed roots are 10 to 100 times more sensitive to auxin presence than normal roots. Because the roots do not exhibit altered, auxin-independent proton excretion, altered ethylene sensitivity, or increased auxin uptake they suggested that the transformation may

0735-2689/91/$.50 O 1991 by CRC Press, Inc.

affect the auxin reception-transduction system. The increased auxin sensitivity induces root induction, which is known to be initiated by auxins, by endogenous auxin concentrations. However, the issue is far from resolved. Croes et alV5 found no differential sensitivity between transformed roots and normal root cultures. Cardarelli et a1 .6

have demonstrated that A. rhizogenes with the rolB plasmid, i.e., pRi 18% with all but ORF 1 1 of the T,-DNA deleted,7 is incapable of initiating roots on carrot discs in the absence of exogenous auxin, The role of auxins was also examined by Quattrochio et a1.8 by supplying the antiauxins 2,4,6-trichlorophenoxyacetic acid, 2,3,5-triio- dobenzoic acid, or p-chlorophenoxyisobutyric acid to potato hairy roots; these inhibited root growth. Ohkawa et aL9 found that GA, was stimulatory to the growth of hairy roots of Datura innoxia, while an inhibitor of gibberellin biosynthesis, (E)- 1 "(4-chloropheny1)-4,4-dimethyl-2- (1,2,4-triazol- 1-y1)- 1 -penten-3-ol(S-3307, was inhibitory. Clearly, further studies of the roles of endogenous hormones in induction and mainte- nance of hairy roots, and optimum levels and mixtures of exogenous hormones, are required.

De Cleene and De Leyt0 surveyed the host range of this bacterium, listing 192 species which had been artificially infected with A. rhizogenes to determine whether successful infection would occur, and also whether infection is a character of taxonomic importance. This work has been criticized on two grounds. Anderson and Moore1 tested "hairy-root" induction by 19 A. rhizogenes strains and found there was little correlation between the strain used and the taxonomic position of the host plant. De Cleene and De Ley also used only one bacterial strain, TR7, and generally only one variety of each possible host for their infection tests, and so did not consider whether strain specificities exist on the part of either the pathogen or the host.

Since 1981, over 300 additional taxa have been tested for susceptibility to A. rhizogenes infection. The reason for the explosion of infection tests has little to do with plant pathology, but, rather, plant biotechnology. The roots produced in the infection can be removed and cultured in simple, hormone-free media where they exhibit a high rate of growth and genetic stability12J3 and high levels of production of sec-

ondary metabolites. 1 4 9 1 5 These transformed root cultures have also been used for studies of my- conhizal fungi, lb18 h e r b i ~ i d e l ~ ? ~ ~ and nematicide21 action and transport in dicots, studies of root- parasitic nematodes bacteria,= and fungi,25*26 examination of uptake of metals from sewage sludges ,27 production of plant enzymes ,28 and the transfer of transposable elements. 29

With the large increase in literature related to A. rhizogenes, it has become time to assess again the value of such information in taxonomic *P

and systematic research, particularly to suggest *;

the most valuable avenues for further research in this area. It is difficult for the worker in the field f

to keep track of the species which have and have not been tried or infected. New workers to this area may find little guidance because of the wide variety of sources in which this information occurs. It is for these groups that this compilation has been prepared. For the sake of completeness, it was decided to include the work of De Cleene and De Ley,1° as well as earlier references which have been seen. While it is the intent in this review to critically examine the host range of A. rhizogenes, the virulence of bacterial strains used in infection trials, factors which may control the expression of symptoms and infection, and the systematic value of this information, no attempt has been made to review the genetics or molecular biology of these bacteria. This area has been recently reviewed. 3y30

If. INFECTION TRIALS

A. Literature Data

e 44. The compilation of infection trials has oc- " I(

curred as a result of studies in this author's laboratory of infection of plants of medicinal value. 4

u4.

Every attempt has been made to be exhaustive in r

regard to taxa (primarily specific and infraspe- cific) and the bacterial strain used, although where duplications occur in the literature (and these are frequent), only one reference is given, usually that listing either the greatest number of species infected or the greatest number of bacterial strains used. From the available information, a micro- computer database has been prepared which includes the species inoculated, variety, if given,

the plant family, bacterial strain(s) used, the results of the inoculation trial, and the pertinent reference. The database is much more exhaustive of the literature than the listing presented here; it contains multiple entries for several taxa de- pending on the number of researchers which have reported work with the plant.

B. Author's Own Studies

For studies of infection carried out in this author's laboratory, strains ATCC 1 1325, ATCC 15834, ATCC 43057, and A4 have been main- tained on either nutrient broth or potato dextrose broth. Generally, it was found more successful to transfer the bacteria to agar culture of the same medium prior to infection. A variety of infection techniques have been used, including flooding a carborundum abraded area with the liquid bacterial culture, injection of a liquid suspension shallowly or deeply into leaf veins or stems with a hypodermic needle, stabbing the plant with a needle or toothpick loaded from a colony, or slicing with a sterile scalpel similarly loaded with bacteria. Although vaseline was sometimes used to close the area of the wound, it was generally found more successful in this laboratory to main- tain the plant at high humidity and to inoculate with a sterile toothpick. Species which have been inoculated in this laboratory include Kalanchoe diagremontiana (a positive control), Coleus blumei, Aloe variegata, Acacia drummondi, Cat- tleya hybrid (Bic. Mem Crispin Rosales X Naomi Kerns XXX SLC. Vallezas "illy Miles' AM/ AOS-Purple), Rebutia sp. , Dionaea muscipula, Ruta graveolens, Oscularia canescens, Sesamum indicum, Gossypium herbaceum, Pisum sativum cv Early Alaska, Antirrhinum majus cv Princess, Phaseolus vulgaris cv Pinto, Crassula argentea, Capsicum frutescens var longum cv Cayenne, Borago officinalis, Solanum sodomeum, S. auriculatum, S. melongena var esculenta, Dicentra spectabilis, Albizia julibrissin, Gaillardia aristata, Cynoglossum amabile, Tropaeolum majus cv Cherry Rose, Viola septentrionalis, Salvia verticillata, Stachys olympica, Saintpaulia ionantha cv Julie, Zingiber oflcinale, Nymphoides aquatica, Iresine herbstii, Fittonia Verschafeltii var argyroneura cv Minima, Lophophora wil-

liamsii (callus), Podophyllum hexandrum ( = P. emodi), and P. peltatum. Some of these plants were obtained locally or purchased from local commercial sources. Others were grown from seed obtained from a commercial source or from the Medicinal Plant Garden, Department of Phar- macognosy, University of Rhode Island (Kings- ton, RI). The callus of Lophophora williarnsii was kindly supplied by William Obemeyer of the Philadelphia College of Pharmacy & Science. In some cases (K. diagremontiana, C. annuum var acuminatum, and P. peltatum), the success of transfer of bacterial DNA to roots or hyper- trophied tissues at the wound was tested by determining the presence of opines using the method of Petit et aL3'

Iff. THE HOST RANGE

A complete compilation is given (Table 1) for plants in which infection by Agrobacterium rhizogenes has been attempted. This table shows the family, species, variety, and results of the trial for 645 subgeneric taxa, including 183 named varieties of 463 species in 109 families, 49 orders, 5 classes, and 2 divisions. For the conven- ience of those who lack formal taxonomic train- ing, this listing is provided alphabetically among and within families. The bacterial strains used for infection in each taxon are indicated, along with the success or failure of that infection trial.

A. Plant Infection with A. rhizogenes

Several conclusions can be drawn from this compilation of plant species and varieties which have been tested for infection with A. rhizogenes. De Cleene and De Ley10 reported that, of 202 dicotyledons tested, only 37 species (18%) were infected. In contrast, Table 1 shows infection in 264 species (57% of those tested). De Cleene and De Ley showed infection in 15 families with con- centration in the Asteridae and Rosidae, while the present report shows susceptible hosts in 49 families distributed in all dicot subclasses. It should be noted that a very large fraction (171 of 206 or 83%) of those which have not been demonstrated to be hosts of A. rhizogenes are

A Compilation of Plant Species Infection Trials with A. rhizogenes

Species

Acant haceae Aphelandra squarrosa Nees. var leopoldii Van Houtte Fittonia Verschafeltii Coem. var argyroneura Nichols. Phaylopsis longifolia Sims Ruellia lorentziana Griseb.

Actinldiceae Actinidia chinensis Planchon

Ag avaceae Dracaena sp. cv. Volckahertii Sanseveria trifasciata Prain var laurentii N.E. Brown

Aizoaceae Oscularia canescens (Mill.) Schwant.

Amaranthaceae Aerva scandens (Roxb.) Wall. Amaranthus retroflexus L. lresine herbstii Hook. f.

Apiaceae Bupleurum ranunculoides L. Coriandrum sativum L. Daucus carota L.

Daucus carota L. cv Chantenay Daucus carota L. cv Nantaise Daucus carota L. cv Nantaise amelioree Daucus carota L. var sativa Daucus carota L. var 82BR Foeniculum vulgare Mill. Heracieum sphondilium L. Pastinaca sativa L. Peucedanum muscarie? Pimpinella anisum L.

Apocynaceae Catharanthus roseus (L.) G. Don Catharanthus roseus (L.) G. Don cv Little Delicata Catharanthus trichophyllus (Bak.) Pich. Ervatamia obtusifolia ? (= Tabernaemontana obtusifolia Poir.) Holarrhena fioribunda Durand & Schinz Strophanthus petersianus Klotzsch

Araceae Caladium bicolor (Ait.) Vent. Philodendron elaphoglossoides Schott x P. wendlandii Schott cv. Lynette

Zantedeschia aethiopica (L.) Spreng. Araliaceae

Panax ginseng C. A. Meyer Aristolochiaceae

Aristolochia baetica L, Asclepiadaceae

Alexitoxicum laxum (Bartl.) Fobed. fide Willis Frerea indica Dalz. Stapelia sp.

Asteraceae Achillea millefolium L.

Result Ref.

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TABLE 1 (continued) A Compilation of Plant Species Infection Trials with A. rhizogenes

Species Result Ref.

Acmella oppositifolia (Lamarck) R. K. Jansen Ambrosia artemisiifolia L. Artemisia absinthium L. Artemisia annua L. Artemisia vulgaris i. Bidens alba L. vat radiata (Schz. Bip.) Ballard Bidens ferulaefolia (Jacq.) DC. Bidens pilosa L. Bidens pilosa L. var minor (Blume) Sherff Bidens sulphureus Sch. Bip. Carthamus tinctorius L. cv Biggs Carthamus tinctorius L. cv N-10 Centaurea cyanus L. Chaenactis douglasii Hook. & Arn. Chrysanthemum frutescens L. Chrysanthemum morifolium (Ramat.) Hemsl. cv White Top Cichorium endiva L. Cichorium intybus L. Coreopsis grandiflora Hogg ex Sweet var harveyana (Gray) Sherff Coreopsis grandiflora Hogg ex Sweet var longipes (Hook.) T. & G. Coreopsis grandiflora Hogg ex Sweet cv Mayfield Giant Coreopsis maritima (Nutt.) Hook. f. Coreopsis nuecensis A. Heller Coreopsis nuecensoides E. B. Smith Coreopsis tinctoria Nutt. var tinctoria Crepis capillaris Nutt. Gaillardia aristata Pursh Gaillardia pulchella Foug. Galinsoga parviflora Cav. Helianthus annuus L. Helianthus tuberosus L. Matricaria charnomilla L. Rudbeckia hirta L. var pulcherrima Saussurea alpina DC. Senecio serpens Rowl. Senecio vulgaris L. Stevia rebaudiana Bertoni Tagetes erecta L. Tagetes patula L. Tagetes patula L. cv Nana

Balsaminaceae lmpatiens balsamina L. lmpatiens capensis Merb. lmpatiens holstii Engler & Warb.

Basellaceae Anredera spicata Pers.

Begoniaceae Begonia manicata Brongn. Begonia richmondensis Hort. Begonia x fuchsifolia Chev.

Berberidaceae Podophyllum hexandrum Royle (= P, emodi Wall.) Podophyllum peltatum L.

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Species

Betulaceae Betula pendula Roth.

Bignoniaceae Campsis grandiflora (Thunb.) K. Schum. Jacaranda mimosaefslia D. Don

Boraginaceae Anchusa officinalis L. Borago officinalis L.

Cynoglossum amabile Stapf & Drummond Lithospermum erythrorhizon Sieb. et Zucc. Myosotis palustris L. Symphytum officinale L.

Brassicaceae Arabidopsis thaliana (L.) Heynn. Arabidopsis thaiiana (L.) Heynn. cv Dijon Arabis perfoliata Lam. Arabis sagittata (Bertol.) DC. Arabis turrita L. Armoracia lapathifolia Gilib. Armoracia rusticana P. Gaert., B. Meyer et Scherb. Brassica campestris L. var napo-brassica DC. Brassica chinensis L. Brassica hirta Moench. Brassica napus L. Brassica napus L. cv Brutor Brassica napus L. cv Jet Neuf Brassica napus L, cv Rafar Brassica napus L. cv Victor Brassica napus L. cv Willi Brassica napus L. var oleifera Metz. cv Darmor Brassica oleracea L. var acephafa cv Shirohato Brassica oleracea L. var botrytis L. Brassica oleracea L. var botrytis L. cv Snow Crown Brassica oleracea L. var capitata L. Brassica pekinensis Rupr. Brassica rapa L. Brassica rapa L. cv Purple Top Brassica spp. Capsella bursa-pastoris (L.) Medik. Cheiranthus scoparius Brouss. Matthiola fruticulosa (L.) Maire Raphanus sativa L. Sinapis alba L. Thlaspi arvensis L.

Cactaceae Echinocactus sp. Lophophora williamsii (Lem.) Coulter Opuntia humifusa Raf. Pereskia aculeata (Plum.) Mill. var rubescens Pfeiff. Pereskiopsis velutina Rose Rebutia sp.

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TABLE 1 (continued) A Compilation of Plant Species Infection Trials with A. rhilogenes

Species

Calycanthaceae Chimonanthus praecox (L.) Link Chimonanthus yunnanensis W. W. Sm.

Campanulaceae Campanula spicata L. Campanula vidalii H. C. Wads, Jasione montana L. Lobelia inflata L.

Capparaceae Maerua angolensis DC.

Caprifoliaceae Lonicera caerula L. Lonicera japonica Thunb. var aureo-reticulata Nichols. Lonicera morrowii A. Gray Lonicera periclymenum L. Lonicera sp. Lonicera tartarica L. Symphoricarpos racemosus Michx.

Caryophyllaceae Dianthus caryophyllus L. Gypsophila fastigiata L. Gypsophila muralis L. Gypsophila repens L. Paronychia argentea Lam. Silene armeria L. Sifene conica L. Silene infiata L. Siiene maritima With. Spergula arvensis L. Stellaria holostea L. Stellaria Media (L.) Vill.

Chenopodiaceae Beta vulgaris L. Beta vulgaris L. cv Boltardy Beta vulgaris L, cv Regina Beta vulgaris L. cv SVP 2425 Beta vulgaris L. var attissima Beta vulgaris L. var conditiva Beta vulgaris L. var crassa Alef Beta vulgaris La var vulgaris Beta vulgaris L. x B, patellaris AN5 Chenopodium rubrum L.

Cistaceae Helianthemum canum (L.) Baumg. Tuberaria guttata (L.) Fourr.

Clusiaceae Hypericum perforaturn L.

Combretaceae Combretum Farinosum H.B.K.

Commelinaceae Callisia elegans Alexander Cornmelina clandestina Mart. Cyanotis kewensis (Hassk.) C.B. Clarke

Ref.

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Species Result Ref.

Convolvulaceae Convolvulus arvensis L. Convolvulus sepium L. (= Calystegia sepium R.Br.) ipomoea aristolochiaefolia L. lpomoea batatas L. lpomoea purpurea (L.) Roth.

Coriariaceae Coriaria terminalis Hemsl.

Crassulaceae Andromischus sp. Crassula argentea Thunb. Crassula rosularis Haw. Crassula tillaea Macl. Echeveria canaliculata Hook. Kalanchoe blossfeldiana V. Poelln. Kalanchoe daigremontiana Ham. & Perrier Kalanchoe pinnata (Lam.) Pers. (= Bryophyllum pinna- turn (L.f.) Kurz; B. calycinum Saiisb.)

Kalanchoe sp. Kalanchoe tubiflora (Harvey) R. Hamet Sedum acris L. Sedum moranense H.B.K. Sedum rubrotinctum R.T. Clausen cv Aurora Sedum spectabile Boreau Sempervivum montanum L.

Cucurbitaceae Benincasa hispida (Thunb.) Cogn. Citrullus vulgaris Schrad. Cucumis Melo L. Cucumis sativus L. Cucumis sativus L. cv Straight 'Eight

Cupressaceae Callitris endlicheri (Parl.) F.M. Bailey

Cyperaceae Cyperus esculentus L.

Diosco reaceae Dioscorea alata L. Dioscorea batatas Decne.

Dipsacaceae Scabiosa leucophylla Borb. Scabiosa rhodopensis Stoy. & Stef. Succisa pratensis Moench

Droseraceae Dionaea muscipula Ellis

Elaeagnaceae Elaeagnus angustifolia L. Efaeagnus commutata Bernh. ex. Rydb.

Elaeocarpaceae Aristotelia australasica

Ericaceae Erica ciliaris L. Erica lusitanica Rud.

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Species

Eu phorbiaceae Acalypha parettii Spreng. Codiaeum variegatum (L.) A. Juss. cv Mme Draps Euphorbia marginata Pursh Euphorbia spp. Euphorbia trigona Haw. Phyllanthus grandifolius L.

Fabaceae Mimosaceae

Acacia drummondi Benth. Acacia gerrardi Benth. Albizia julibrissin Durazz. Ceratonia siliqua L.

Caesalpinaceae Cassia obtusifolia L. cv Ketsumeishi Cassia occidentalis L. Cassia torosa Cav. Gleditsia triacanthos L.

Papilionaceae Abrus precatorius L. Arachis hypogaea L. Caragana arborescens Lam. Glycine canescens F.J. Hermann Accession GI 11 4 Glycine canescens F.J. Hermann Accession GI 171 Glycine canescens F.J. Hermann Accession GI 240 Glycine canescens F.J. Hermann Accession GI249 Glycine canescens F.J. Hermann Accession GI340 Glycine canescens F.J. Hermann Accession GI 699 Glycine canescens F.J. Hermann PI 399478 Glycine clandestina Wendl. Accession G 1001 Glycine clandestina Wendl. Accession GI 01 9 Glycine clandestina Wendl. Accession G I 145 Glycine clandestina Wendl. Accession GI231 Glycine rnax (L.) Merr. Glycine max (L.) Merr. cv Arksoy Glycine rnax (L.) Merr. cv Beeson Glycine rnax (L.) Merr. cv Biloxi Glycine rnax (L.) Merr. cv Bragg Glycine rnax (L.) Merr. cv Cartter Giycine max (L.) Merr. cv Clark Giycine rnax (L.) Merr, cv Clark rji Glycine rnax (L.) Merr. cv Cobb Glycine max (L.) Merr. cv Coker 102 Glycine max (L.) Merr. cv Columbia Glycine max (L.) Merr. cv Corsoy 79 Glycine rnax (L.) Merr. cv Cutler 71 Glycine rnax (L.) Merr. cv Emerald Giycine rnax (L.) Merr. cv Essex Glycine rnax (L.) Merr. cv Fayette Glycine max (L.) Merr. cv Fiskeby V Glycine rnax (L.) Merr. cv Forrest Glycine rnax (L.) Merr. cv Franklin Glycine max (L.) Merr. cv Govan

Result Ref.

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TABLE I (continued) A Compilation of Plant Species Infection Trials with A. rhizogenes

Species

Glycine rnax (L.) Merr. cv Hampton Glycine rnax (L.) Merr. cv Jupiter Glycine rnax (L.) Merr. cv Kanrich Glycine rnax (L.) Merr. cv Kent Glycine rnax (L.) Merr. cv Lee Glycine rnax (L.) Merr. cv Manchu Glycine rnax (L.) Merr. cv Mandarin Glycine rnax (L.) Merr. cv Maple Arrow Glycine rnax (L.) Merr. cv Mitchell Glycine rnax (L.) Merr, cv Peking Glycine rnax (L.) Merr. cv Pickett Glycine rnax (L.) Merr. PI 398.479 Glycine rnax (L.) Merr. PI 398.580 Glycine rnax (L.) Merr. PI 398.61 1 Glycine rnax (C.) Merr. cv Ransom Glycine rnax (L.) Merr. cv Richland Glycine rnax (L.) Merr. cv Seminole Glycine rnax (L.) Merr. cv Seneca Glycine rnax (L.) Merr. cv Sooty Glycine rnax (L.) Merr. cv Verde Glycine rnax (L.) Merr. cv Williams Glycine rnax (L.) Merr. cv Williams 82 Glycine rnax (L.) Merr. cv Wing Jet Glycine soja Sieb. & Zucc. Glycine soja Sieb. & Zucc. PI 342.434 Glycine soja Sieb. & Zucc. PI 378.6938 Glycine soja Sieb. & Zucc. PI 407.287 Glycine soja Sieb. & Zucc. PI 65549 Glycine soja Sieb. & Zucc. PI 81762 Glycyrrhiza glabra L. Glycyrrhiza uralensis Fisch. ex DC. Lathyrus vernus (L.) Bernh. Lotus cornicuiatus L. Lotus corniculatus L. cv Rodeo Luburnum alpinum (Mill.) Bercht. & J.S. Presl. Lupinus albus L. Lupinus polyphyllus Lindl. Macroptilium atropurpureum L. Medicago sativa L. Medicago sativa L. cv Adriana Medicago sativa L. cv Europe Medicago sativa L. cv Hunter River Medicago sativa L. cv Ladak 65 Medicago sativa L. cv Vernema Medicago tornata Webb, ex Bail Phaseolus lunatus L. breeding line L126 Phaseolus vulgaris L. Phaseolus vulgaris L. cv Blue Lake Phaseolus vulgaris L. cv Pinto Pisum sativum Pisum sativum L. cv 'Nain tres hatif dlAnnonay', Truffaut Pisum sativum L. cv Early Alaska Pisum sativum L. ssp arvense


Species Result

Psophocarpus tetragonolobus DC. Robinia pseudoacacia L. Sesbania rostrata Brem. Sophora tetraptera J. Mill. Stylosanthes humilis HB&K cv Paterson Trifolium fragiderum L. Trifolium pratense L. Trifolium repens L. Trifolium sp. Trifolium subterraneum L. Vicia faba L. Vicia faba L. cv Fribo Vicia faba L. cv Windsor Vicia faba L. var equina cv H51/3 Vicia faba L. var equina cv Sudanese Triple White Vicia faba L. var equina cv Blandine Vicia faba L. var major cv Optica Vicia faba L. var major cv 859 Vicia faba L. var minor cv BPL 1192 Vicia faba L. var minor cv SCI Vicia faba L. var paucifuga Vicia sativa L. Vigna aconitifolia (Jacq.) Marechal Vigna unguiculata L.

Fumariaceae Dicentra spectabilis Lem.

Funariaceae Funaria hygrometrica Hedw.

Gentianaceae Gentiana asclepiadea L. Gentiana cruciata L. Gentiana lutea L. Swertia japonica Makino

Geraniaceae Geranium molle L. Geranium Robertianum L. Pelargonium hortorum Bailey cv Irene Pelargonium hortorum Bailey cv Penny Pelargonium hortorum Bailey cv Radio Red Pelargonium sp. Pelargonium zonale Hort.

Gesneriaceae Chirita lavandulacea Stapf Episcea dianthiflora H. E, Moore & R.G. Wils. Episcea lilacina Hanst. Kohleria bogotensis (Nichols.) Fritsch Saintpaulia ionantha H. Wendl. Saintpaulia ionantha H. Wendl. cv Julie Sinningia speciosa Benth. & Hook. Tussacia friedrichstahliana Hanst.

Glo bulariaceae Globularia cordifolia L.

Ref.

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Species

Grossulariaceae Ribes petraeum Wulf.

Hypnaceae Pylaisiella selwynii (Kindb.) Crum

Juglandaceae Juglans sp.

Juncaceae Luzula spicata DC.

Lamiaceae Coleus arabica Benth. Coleus blumei Benth. Lamium album L. Mentha aquatica L. Salvia sclaraeoides Bort. Salvia verbenaca L. Salvia verticillata L. Stachys nivea (Stev.) Benth. Stachys olympica Poir. Teucrium rnassiliense L.

Liliaceae Aloe striatula Haw. Aloe tenuior Haw. Aloe variegata L. Asparagus aethiopicus L. Bulbine semibarbata (R.Br.) Haw.

Linaceae Linum grandiflorum Desf. Linum usitatissimum L.

Loasaceae Blumenbachia hieronymi Urb.

Malvaceae Abutilon theophrasti Medick. Gossypium herbaceurn L. Hibiscus esculentus L.

Marantaceae Calathea sp.

Marcgraviaceae Marcgravia polyantha Delp.

Melastomataceae Bertolonia marmorata Naud, cv Bruxelliensis Tibouchina paratropica Cogn.

Menispermaceae Cocculus trilobus (Thunb.) DC.

Menyanthaceae Nymphoides aquatica (Walt.) Ktze.

Moraceae Dorstenia contrajerva L. Morus alba L.

Myoporaceae Bontia daphnoides L.

M yrsinaceae Ardisia crenata Sims Ardisia humilis Vahf

Result Ref.

- e 10 - e 10

kq, -m This paper - 9 10 - e 10

- e 10 - m This paper +" 103, this

paper

- m This paper


Species

M yrtaceae Eucalyptus gunnii Hook. f. Eugenia balsamea Wight Tristania conferta R.Br.

Nyctaginaceae Boerhavia repens L.

Oleaceae Armeria nebrodensis Guss. Fraxinus americana L. Jasminum fructicans L. Oiea europaea L. Ofea europaea L. cv Manzenilo

Onagraceae Oenothera biennis L.

Orchidaceae Cattleya hybrid

Oxalidaceae Oxalis ortgiesii Regal

Papaveraceae Chelidonium majus L. Papaver somniferum L. Sanguinaria canadensis L.

Pedaliaceae Sesamum indicum L.

Phytolaccaceae Phytolacca dioica L. Rivina humilis L.

Pinaceae Abies procera Rehd. Larix decidua Mill. Picea glauca (Moench) Voss Pinus ponderosa Pseudotsuga menziesii Tsuga heterophylla Sarg.

Piperaceae Peperomia cumbreana Trelease & Yunker

Plantaginaceae Plantago lanceolata L. Plantago major L.

Plumbaginaceae Armeria majellensis Boiss.

Poaceae Hordeum vulgare L. cv Maris Otter Panicum miliaceum L. Triticum aestivum L. cv Norman Zea mays L, cv Golden Cross Bantam

Pol ygonaceae Oxyria digyna (L.) Hill Polygonum aviculare L. Polygonum convolvulus L. Polygonum hydropiper L. Rheum palmatum L. Rumex acetoseila L.

Result Ref.

- m This paper

- e 10 + cegku 79

- c This paper

+ c This paper

+ cdegux - w 9 40 + 39 - u 4 1

+ cdeg - uwx 1 40

+ cdegux - w 9 40

+ cdgx9 - euw 40


Species

Rumex crispus L. Rumex obtusifolius L.

Polypodiaceae Cyrtomium falcatum (L.f.) K.B. Presl. Dennstaedtia cicutaria (Sw.) Kuhn Pdyp~dium phyllitidis L,

Primulaceae Anagallis arvensis L. Lysimachia vulgaris L. Primula x Kewensis W. Wats.

Ranunculaceae Aconitum barbutum Patr. ex Pers. Aquilegia alpina L. Aquilegia canadensis L. Delphinium elatum L. Delphinium orientale J. Gray Delphinium sp. Delphinium zalil Aitsch. & Hemsi. Ranunculus repens L.

Resedaceae Reseda media Lag.

Rosaceae Cotoneaster acuminatus Lindl. Cotoneaster harrysmithii Flinck & Hylmo Cotoneaster parneyi Hort. Cydonia oblonga Mill. Geum reptans L. Geum urbanum L. Malus domestica Baumg. Malus floribunda Sieb. Malus pumila Mill. cv Greensleeves Malus pumila Mills. cv Tuscan Malus pumila Mill. cv M.9 Malus pumila Mill. cv M.25 Malus sp. Malus Sylvestris Mill. Malus sylvestris Mill. cv Delicious Potentilla bifurca L. Potentilla erecta (L.) Raeusch Potentilla recta L. Prunus amygdalus Batsch cv Amandier Amer Prunus amygdalus Batsch cv #51 grafted on cv #14 (Israeli cvs) Prunus domestica L. cv Myrobolan Prunus persica Sieb. & Zucc. (non Batsch) Prunus persica Sieb. & Zucc. (non Batsch) cv Missour Pyrus communis L. cv Bartlett Rosa multiflora Thunb. Rosa multiflora Thunb. ex Murr var japonica Rosa setigera Michx. Rosa sp. Rubus allegheniensis T. Porter cv Darrow Rubus idaeus L. Rubus runssorensis Engl.

Ref.


Species

Rubus ursinus Cham. & Schlecht. var loganobaccus Bailey Rubus X neglectus C.H. Peck cv Royalty Spiraea prunifolia Sieb. & Zucc. Spiraea sp. Spiraea X vanhouttei (Briot) Zab.

Rublaceae Cinchona ledgeriana L. Coffea arnoldiana De Wild. Galium aparine L. Gardenia jasminoides Ellis

Rutaceae Ruta graveolens L.

Salicaceae Populus davidiana Dode Populus tremuia L. x P. alba L. Populus trichocarpa Torr. & Gray ex Hook. x P. deltoides Marsh.

Saxif ragaceae Saxifraga rotundifolia L.

Scrophulariacae Antirrhinum majus L. Antirrhinum majus L, cv Princess Antirrhinum meoanthum Hoffmg. & Link Calceolaria chelidonioides H.B.K. Calceolaria nodosa L. Digitalis lanata Ehrh. Digitalis purpurea L.

Verbascum lychnitis L. Simarou baceae

Ailanthus altissima (Mill.) Swingle Ailanthus vilmoriniana Dode

Solanaceae Atropa belladonna L. Atropa caucasica Kreyer Capsicum frutescens L. Capsicum frutescens L. var longum Bailey cv Cayenne Cestrum parqui I' Her. Datura candida Saff. X D. aurea Saff. Datura chlorantha Hook. Datura fastuosa L, var violacea Datura ferox L. Datura innoxia Mill. Datura metel L. Datura meteloides DC. ex Dun. Datura quercifolia H.B.K. Datura rosei Saff. Datura sanguinea Ruiz. et Pav. Datura sp. Datura stramonium L. Datura stramonium L. var inermis Timm. Datura stramonium L. var stramonium Dun. Datura stramonium L. var tatula Torr. Duboisia hybrid

Result Ref.

65 140 137 74

137

105 10 10 10

This paper

141 27 92

10

103 This paper

10 10 10 27

142, this paper

10

10 107

103 143 142

This paper 10

144 143 143 143 143 143 143 143 143 143 145 103 143 143 143 143


Species

Duboisia leichhardtii F. Muell. Duboisia myoporoides R.Br. Hyoscyamus albus L. Hyoscyamus aureus L. Hyoscyamus bohemicus F.W. Schmidt Hyoscyamus muticus L. Hyoscyamus niger L. Hyoscyamus niger L. var pallidus Rchb. Hyoscyamus turcomanicus Pojark. Lycopersicon esculentum Mill. Lycopersicon esculentum Mill. cv Earliana Lycopersicon esculentum Mill. cv Marmande Lycopersicon esculentum Mill. cv Pearson Lycopersicon esculentum Mill cv Rutgers Lycopersicon esculentum Mill. cv South Australian Early Dwarf Red Lycopersicon esculentum Mill. cv Tropic Lycopersicon lycopersicum (L,) Farw. Lycoperison peruvianum Mill. Nicotiana africana Memmuller Nicotiana benthemiana Domin Nicotiana cavicola Burbidge Nicotiana debneyi Domin Nicotiana cavicola Burbidge Nicotiana debneyi Domin Nicotiana glauca Graham Nicotiana glauca Graham cv Smith Nicotiana hesperis Burbidge Nicotiana occidentalis Wheeler Nicotiana plumbaginifolia Viviani Nicotiana rustica L. Nicotiana rustica L. cv V12 Nicotiana tabacum L. Nicotiana tabacum L. cv Gatersleben Nicotiana tabacum L. cv LAFC Nicotiana tabacum L. cv Mamont Nicotiana tabacum L. cv Petite Havana Nicotiana tabacum L. cv SC58 Nicotiana tabacum L. cv White Burley Nicotiana tabacum L. cv Wisconsin 38 Nicotiana tabacum 1. cv Xanthi Nicotiana umbratica Burbidge Nicotiana velutina Wheeler Petunia hybrida Vilm. Physalis pubescens L. Scopolia anornala (Link 81 Otto) Airy-Shaw Scopolia carniolica Jacq. Scopolia japonica Kuntze Scopolia lurida ? (= S. stramonifolia (Roxb.) N. P. Balakrishnan) Scopolia straminifolia (Wall.) Shrestha Solanum aculeatissimum Jacq. Solanum auriculatum Ait. Solanum dulcamara L. Solanum laciniatum Ait.

Result

+ Cp +@, - '

+ "IUY

S C S C

+ acglmp, - no + "I~PUY

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Ref.

145 147 143 143 143 146 146 143 1 48 103 65 31 65

149 149 149 137 150 151 90

151 90

151 90

152 153 151 90 90

146 151 146 151 151 1 54 155 151 156 157 10

151 151 158 10

146 143 146 159 143 10

This paper 90 15


Species

Solanum lanatum Dun. Solanum melongena L. var esculentum Nees. Solanum nigrum L. Solanum sisymbriifolium Lam. Solanum sodomeum L. Solanum tuberosum h. Solanum tuberosum L. cv Bintje Solanum tuberosum L. cv Desiree Solanum tuberosum L. PDQ07 Solanum tuberosum L. cv Roseval Solanum tuberosum L. cv Zvikov

Sterculiaceae Cola quinqueloba Garcke

Taccaceae Tacca chantrieri Andre

Thymelaceae Daphne gnidium L.

Tropaeo I aceae Tropaeolum majus L. cv Cherry Rose

Ulrnaceae Ulmus americana L.

Urticaceae Pellionia repens (Lour.) Merr. Pilea spruceana Wedd. Urtica urens L.

Valerianaceae Valerianella locusta (L.) Betcke

Verbenaceae Clerodendron serratum Spreng. Verbena rigida Spreng.

Violaceae Viola septentrionalis Greene Viola tricolor L.

Vitaceae Cyphostemma gigantophyllum (Gilg & Brandt) Descoings Vitis (Seneca x NY 45910 [Bath x Interlaken]) cv Vanessa Seedless Vitis L. hybrid cv Remaily Seedless Vitis vinifera L. cv Grenache

I, Zingi beraceae Zingiber officinale Roscoe

Zygophyllaceae Guaiacum sanctum L. Peganum harmala L.

Ref.

27 This paper

90 27

This paper 90 13

160 161 31

162

10

10

10

This paper

122

10 10 10

103

10 10

This paper 10

10 1 40 1 40 163

103, this paper

10 164

Note: Within this table, + = the taxon is infected; - = no successful infection has been reported; +- = infection may occur but has not been confirmed (no root production). Where species could not be confirmed by reference to Index Kewensis, the binomial is followed by ?. Superscript letters following each result indicate the strains used in the infection trial: a unknown; wild type; A4;

K47; ICPB TR7; ICPB TRIO1 ; Q ICPB TR105; 232; ICPB TRI 07; 1 NCPPB 1855; NCPPB 2655; NCPPB 81 96; ATCC 1 1325; ATCC 13332; o ATCC 13333; ATCC 15834; ATCC 43057;

K599; HRI; BL3 - ; C58Ci a; C58Cl b; C58Cl c; C58Cl d; A41ARCx.

from the De Cleene and De Leyt0 report. Their work was carried out with the bacterial strain TR7, about which more will be discussed later.

Some dicot families presented in this report are particularly notable. At the time of the work of De Cleene and De Ley, the Rosaceae and Asteraceae were recognized to contain the most likely susceptible host plants. This has led to considerably more work in the Asteraceae for the production of the secondary metabolites of this group14 using hairy root infections. Until recently, very little additional work had been done in the Rosaceae. This family has now become the focus of experiments to increase the rooting of nursery stock by hairy root infection to promote greater survival during transplanting. 32733

Several dicot families, based on the work of De Cleene and De Ley and that of others, have been considered relatively resistant until recently. These include the Poiygonaceae , Cary- ophyllaceae , Brassicaceae , Cucurbitaceae, Con- volvulaceae , and the Scrophulariaceae. These all had been judged to be non-hosts by De Cleene and De Ley.1° These families can now be recognized as susceptible to infection with successful infections in 88, 42, 75, 75, 100, and 43% of those species tested, respectively, Some families which had been recognized as hosts, in some cases in relatively isolated species, have also withstood further analysis. Additional infection trials in the Rosaceae have increased the infection percentage from 65 to 76%, in the Fabaceae (sensu Cronquisr4) from 67 to 9 1 % (the Mimosaceae and Caesalpinaceae, considered separate families, have been infected in 25 and 100% of those tested, respectively), in the Apiaceae from 25 to 63%, and in the Solanaceae from 25 to 93%, compared to the data of De Cleene and De Ley. l o

It should be noted that in the majority of families for which there are data, still relatively few of the species have been tested and most of the species originally tested and found to be negative by De Cleene and De Ley have not been retested. The conclusion of De Cleene and De Ley that the Crassulaceae and Caprifoliaceae also belong in the group of highly susceptible families is still valid since they are infected in 83 and 60% of those which have been tested, respectively. The Caprifoliaceae , however, have been little studied since 198 1; the only negative reports for the

Crassulaceae come from the De Cleene and De Leyi0 work.

The Solanaceae should receive special notice since it has become the most frequently infected and studied plant family. De Cleene and De Ley reported on nine infection trials. The only infections reported were those accomplished by earlier workers; all directly inoculated plants were negative. This leaves the impression that this family is only moderately susceptible, at best; this conclusion cannot be supported today. Over 280 trials of solanaceous species have been reported in the literature with various A. rhizogenes strains. The vast majority of these (including numerous varieties of several of the species) have been successful infection attempts, with only 4 of 57 species tested having proved negative. Three of these, Cestrum parqui I'Her., Physalis pubescens, and * Solanum aculeatissimum Jacq . , were reported in the De Cleene and De Ley paper, while the fourth, Solanum auriculatum Ait., was tested in this laboratory. The Solanaceae has become the most frequently infected and studied plant family. Atropa belladona L. is second perhaps only to Daucus carota L. in the number of reports dem- onstrating successful infection. The former, however, has been used primarily to study alkaloid production in transformed roots while the latter is often used as a positive control. De Cleene and De Leylo reported Atropa belladonna as nonsusceptible !

De Cleene and De Leyl0 also felt that primitive dicots are resistant to infection by A. rhizogenes. Members of the Magnoliidae and Dilleniidae have been further examined leaving little support for this view. Within the Magno- liidae , species within the Ranunculaceae, Papav- eraceae , and Fumariaceae have been successfully infected. In the Dilleniidae, species of the Elaeo- carpaceae, Malvaceae, and Cucurbitaceae are also susceptible. In both of these groups, further work is required; a majority of the species examined have been those inoculated by De Cleene and De Ley.

The monocots, as a whole, also cannot be considered to be resistant to infection. All but two of the monocot subclasses have susceptible species. No susceptible plants have yet been found in the Arecidae and no member of the Alisma- tidae has yet been tested. Of the Liliopsida, 12

families have been examined with 3, the Poaceae, Zingiberaceae, and Dioscoreaceae, proving susceptible. However, there is continuing contro- versy over the susceptibility of monocots to agro- bacterial infection and the utility of agrobacteria in studying the monocots. This situation may occur for one of two reasons. The first is the simple possibility that monocots are not generally susceptible. The second, suggested by the work in the Poaceae, is that, even though gene transfer occurs, the products of the bacterial genes do not promote rooting by the plant cell. Evidence of gene transfer in these cases comes primarily from DNA hybridization experiment^,^^^^^ a technique which has been used more frequently recently but has not been used in most cases in which the species inoculated has been assessed as being resistant, including other monocots. It may also be possible to detect gene transfer based on the morphology of the wound site. One species inoculated in this laboratory, Aloe variegata L., is indicated in Table 1 as 2 , indicating uncertainty in the infection status. No roots were produced as a result of infection with A. rhizogenes strains ATCC 1 1325 and ATCC 43057. The former gave no indication that transformation had occurred. However, within a few weeks of inoculation with the second strain, the wound sites became raised, brown pustules similar to those reported on A. tumefaciens inoculation of Narcissus cv Paper- white (Amaryllidaceae) and Chlorophyturn ca- pense (Liliaceae) . 37 In this author's experience, this type of wound response is very atypical of the wound response, either with or without A. rhizogenes present, and would suggest an interaction between the plant and bacterium. These wounds have not yet been analyzed for opine synthesis or DNA incorporation. A more thor- ough examination for the evidence of plant transformation to include, in addition to root or tumor induction, opine synthesis and DNA transfer may reveal a much more general infection of monocot species.

The possibility that monocots do not produce substances stimulatory to chemotaxis and Vir regulon induction has been recently examined. g"

The ability of plant cell exudates from five monocotyledonous plants to induce activation in an Agrobacterium tumefaciens vir:: lac tester strain increased in the order Hordeum vulgare < Oryza

sativa = Zea mays < Asparagus oficinalis < Triticum monococcum. The stimulatory molecule from wheat was identified as ethyl ferulate and was measured to have a greater inductive activity than acetosyringone. These authors concluded that the inability of agrobacteria to infect monocots must reside in the transfer of the T-DNA, rather than in the induction process.

De Cleene and De Ley1' state that primitive vascular plants (ferns, gymnosperms) are resistant to infection. No infections were shown in any of these groups. This conclusion also does not survive the present analysis. Although the study of De Cleene and De Ley surveyed three genera of ferns in the Polypodiaceae and no one has yet investigated this or any other fern group further, five gymnosperms (Abies procera, Larix decidua, Pinus ponderosa, Pseudotsuga menziesii, and Tsuga heterophylla) have been successfully i n f e ~ t e d . ~ ~ ~ ~ ~ Only Picea glauca (Pinaceae) and Callitris endlicheri (Cupressaceae) remain as being uninfected. 'Op4'

It would also be hard to support a conclusion that only the relatively advanced, vascular plants are susceptible. One moss, Pylaisiella selwynii, has demonstrated s~sceptibil i ty.~~ This is a very interesting finding, which was explained as a response related to the effects of cytokinins in different moss groups. However, except for a negative report for Funaria hygrometrica in the same study, no further work has been done on non- vascular plants.

B. Relation to Chemical Constituents

It is still important to use the data to examine the relationship between infection and the factors which control that infection. A number of compounds, primarily phenolics, are known to play a role in chemotaxis and induction of the bacterium. Others, components of the cell wall or middle lamella, may be important for the initial binding of the bacterium to the cell prior to gene transfer. Still other compounds may play a role, either as inductive or inhibitory agents. The accumulation of organic acids, betalains, anthocyanins, proanthocyanins, ellagic acid, alkaloids, terpenoids, saponins, cyanogenic compounds, iridoids, orobanchin, calcium oxa-

late, phytoecdysone activity, and whether the family is tanniniferous or woody was surveyed for the families being studied from the information of Cronquist ," H e g n a ~ e r , ~ ~ and Sporne ." The presence of various phenolic compounds known to induce infective activity of agro- b a ~ t e r i a ~ ~ t ~ ~ f ~ ~ was also surveyed in H e g n a ~ e r . ~ ~ Plant gum types were recorded based on the information of Stephen.47 These various characters were examined at the family level rather than for genera or species because the information was available for all of the families in which there was an interest, but much less so for subfamilial taxa. Because of the potential to inhibit bacterial activity when in contact with a specific plant, the presence of antimicrobial compounds was surveyed at the species level based on the information provided in McCleary and Walkington ,48 Smith and R ~ i a , ~ ~ and key references in the latter which provided information on a wide variety of specie^.^*-^^ The presence or absence of activity against Gram-negative bacteria (or, in one report, against A. tumefaciens) was recorded for 178 species.

The conclusion of De Cleene and De Leyio that those families which are known to accu- mulate appreciable levels of polyphenolics were more likely to be infected by A. rhizogenes was a predictor of the current understanding of the induction of agrobacteria for plant infection. Ashby et a1.,45 Spencer and Towers,46 and Stachel et aV7 reported on the effect of various phenolic compounds on the induction of the Vir regulon of A. tumefaciens. Collectively, they reported positive induction by sinapyl alcohol, coniferyl alcohol, sinapic acid, sinapic acid methyl ester, sinapinic acid, syringic acid, s yringaldehyde, p- hydroxybenzoic acid, vanillalacetone, vanillin, ferulic acid, ferulic acid methyl ester, 5-hydroxy- ferulic acid methyl ester, 2' ,4' ,4-tri-hydroxy-33- dimethoxy chalcone, and the 3-methoxy chalcone. Ashby et also reported that some of these compounds also induce chemotaxis in agrobacteria. Of these compounds, vanillin, p-hy- droxybenzaldehyde, s yringaldehyde , s yringic acid, ferulic acid, and sinapic acid are apparently ubiquitous in the angiosperm^,^^ either as pre- cursors to lignin or general phenolic compounds. Vanillin and p-hydroxybenzoic acid are ubiqui-

tous in the Gymnosperms as lignin components. These phenolics are therefore likely to be of little value as determinative compounds to explain the presence or absence of infection. Of the remain- der, only enough information could be found for the families of interest for sinapinic acid. There was a slight, but insignificant (a > 0.9), increase in infection in those families known to accu- mulate sinapinic acid compared to the ratio of infected to uninfected plant families overall.

For the other secondary compounds examined, there was no correlation between infection and the presence of tannins, proanthocyanins, organic acids, anthocyanins, ellagic acid, aka- loids , terpenoids, saponins, cy anogenic compounds, iridoids , orobanchin, calcium oxalate , phytoecdysones, woodiness, or gum type, although comparatively little information was available on the last, Again, it is surprising that the phenolic substances (tannins, proanthocyanins, anthocyanins, ellagic acid) should not be more indicative of infectivity given the positive effect of such compounds in infection trials. 57 There was a slight, but insignificant (a < 0.25), negative correlation between betalain accumulation and infection. The only significant correlation (a < 0.0 1) was found with the antibacterial activities; those species which lack activity against Gram-negative bacteria had more infections than would be expected by chance. However, this relationship is complex. The largest group was 83 species which do not exhibit antibacterial activity and are infected by A. rhizogenes, but the next largest group (47 species) was those which are both infected and exhibit antibacterial activity. The group of species which do not exhibit activity and are also not infected is approximately twice as large (31 species) as those which exhibit activity and are not infected (17 species), the smallest group. A clear relationship would predict a much different pattern, i.e., if antibacterial activity were a major controlling factor, it would be expected that the number of species which both exhibit activity and are susceptible to infection would be the smallest group.

Data were also recorded for the accurnulation of anthraquinones , naphthaquinones , polyacetylenes , potassium nitrate, and unusual storage sac- charides (stachyose, lychnose, umbelliferose,

etc.), but there was too little information available or the compounds were too narrowly distributed to attempt pattern analysis.

An alternative approach in determining a pattern to the presence or absence of infection is to exarnine those species which are not infected after a number of trials. It is important to distinguish between those families which have not shown infection when tested in only one species with only a single bacterial strain and those which remain uninfected after attempts in several to many species with a variety of strains; the former group has not been sufficiently studied. For this study, it was decided to consider a family uninfected if five or more species had been examined, preferably in different genera, by inoculations with two or more bacterial strains. The only families which meet these criteria are the Cactaceae, Gesneriaceae and the Liliaceae (with the possible exception already noted). The Lam- iaceae are also included in this category because only one of ten species has consistently, if ever, shown infection, Coleus blumei. These families were reviewed more extensively in C r o n q ~ i s t ~ ~ and H e g n a ~ e r , ~ ~ although incomplete information was available for the various compounds other than those already mentioned. For comparison, two readily infected families, the Ro- saceae and Solanaceae, were included.

Three characters seem to link the resistant families, the general lack of tanniniferous substances, including proanthocyanins and ellagic acid, lack of cyanogenic compounds, and the presence of free or crystallized calcium oxalate. The last is of no special interest because this state is shared with most of the other families, whether infected or not. These characters set these families apart from the Rosaceae, but are shared with the Solanaceae, and so are of apparently little value in elucidating the resistance to agrobacteria. Except for the Cactaceae, these families lack antibacterial substances, or at least no reports have suggested occurrence of such activity in species of these families. This is quite contrary to what might be expected based on the relationship between antibacterial activity and infection noted above. The Rosaceae and Solanaceae also rarely exhibit antibacterial activities. With relatively few exceptions, the four resistant families are principally herbaceous in habit while the Ro-

saceae and Solanaceae contain a number of woody members (in the Solanaceae, mostly herbs have been infected to date). It is possible that the pre- cursors or degree of lignifkation, or perhaps more importantly, the breakdown products on wounding may yet indicate the biochemical basis of A, rhizogenes resistance.

The pattern for A, tumefaciens infection, and so the basis for that pattern, may be slightly different. Some members of the Lifiaceae (Aloe, Asparagus) are susceptible to infection by A. tumefa~iens,~~@ but a great number are resistant to infection by this pathogen as Some members of the Cactaceae, Lamiaceae and Gesneriaceae are susceptible to A. tumefaciens infection. 61 This might indicate some differences in infection capabilities between the two agro- bacterial species, as has been suggested frequently in the literature. However, except for Pereskia ltculeata (Cactaceae) , Episcia dianthiflora, and Kohleria bogotensis (Gesneriaceae), those species found to be resistant to infection with A. rhizogenes are also resistant to A. tumefaciens, These data also point to the need for further work in this area.

The perception that A. tumefaciens is more virulent and has a broader host range has largely dominated research approaches since 1981. This perception should become the focus of further investigation considering the evidence of Table 1 and the fact that a greater number of species have been tested for A. tumefaciens infection. Furthermore, the observation by Marks et al.36 that one A. rhizogenes strain gave better agroinfection of wheat varieties than any of eight strains of A. tumefaciens and one other was equal to the best of the A. tumefaciens strains suggests that studies should always include at least one A. rhizogenes strain.

One further factor should be considered in future analyses of host range in these bacteria. For proper analysis of biosystematic relationships, it is just as important to have negative data as positive. A survey of the majority of the literature on infection trials with both A. rhizogenes and A. tumefaciens suggests that nearly every new species tried is susceptible to infection. Based on the results obtained in this author's laboratory, it is unlikely that this is true. It would therefore seem that there is a wealth of negative data which

has not been reported in the literature. Failure to make this information available not only wastes the time of other researchers in unknowingly re- peating experiments, but also hampers complete analysis of the relationships which exist among the hosts and these bacteria.

C. Analysis of

While single characters may be valuable in the analysis of interspecies interactions if the relationships are simple, analysis of more complex interactions requires the consideration of multiple characters. This is basically the premise of nu- merical taxonomy and can equally be applied here. This has been preliminarily examined here through cluster analysis of the families for which infection data are available based on the 19 characters listed above. The data obtained from C r o n q ~ i s t ~ ~ and HegnaueF3 were given four character states, corresponding to (1) always or generally present, (2) sometimes present, (3) rarely present, or (4) absent. Data from SporneM was less complete and the characters were designated with three character states as (1) always present, (2) sometimes present, and (3) always absent. The cautions discussed by this last author concerning the negative nature of such designations are appropriate here since not all, and in many cases not even a significant number, of the species of a family have been examined.

The cluster analysis was carried out using the UPGMA method,62 which determines mean differences between clusters, using the SPSS/PC + program. The major limitation has been the absence of data for most of the characters in a great number of families. For this reason, the cluster- ing has only been carried out for the dicotyledonous families using the tanniniferous , proan- thocyanin, ellagic acid, and woodiness characters . Again, these characters might be decided a priori to have a significant influence on infection, given the influence of phenolic substances on the Agro- bacterium infection process.

This analysis resulted in the identification of 18 clusters from the 86 dicotyledonous families for which complete data were available. These clusters, containing 2 to 21 families each, were split into 2 major groups, the larger consisting

of 5 1 families and the smaller of 35. This analysis did separate the three dicotyledonous families which have been considered here as sufficiently tested to be considered resistant (Lamiaceae, Cactaceae, and Gesneriaceae in the larger group) from some of the major susceptible families (Crassulaceae, Fabaceae, and Rosaceae in the smaller group). However, both the Lamiaceae and the Cactaceae were clustered together, and equidistant, from the Asteraceae, Convolvula- ceae, Solanaceae, and the Brassicaceae. The Gesneriaceae were grouped, in a cluster one link- age unit more distant, with the Acanthaceae, Zygophyllaceae , Apiaceae , and Linaceae. The larger main cluster had nearly equal numbers of infected and noninfected families (26 and 25, respectively ) , while the smaller cluster had slightly more resistant families (20 vs . 15). This approach has also failed to identify those factors which are most responsible for resistance or susceptibility.

A second analysis involved examining the index developed by SporneM as a measure of advancement of the family based on the presence of primitive or advanced characters. The index was determined by Sporne from data on the presence and absence of 30 characters selected from a list of 107 on the basis of statistical association and was calculated as a percentage advancement index from the ratio of the number of advanced characters present to the total number of characters for which data were known. The values of the index for the extant dicotyledonous families range from 23 for the primitive Degeneriaceae and Aextoxicaceae to 87 for the Dipsacaceae. Two analyses were performed: (1) the frequency distributions of the number of infected, resistant, and all families over the range of the index were tested for fit to a Poisson distribution; and (2) the mean of each distribution of tested families was compared to the mean of all families to test for bias toward more primitive or more advanced families than would be expected by random chance.

All three frequency distributions were visibly skewed to the right, i.e., there were more families at high index values than would be expected based on a normal distribution and the mean was shifted toward values indicating primitiveness. When each distribution was fitted to a Poisson distribution and the significance of the skew (g,)

was e ~ a l u a t e d , ~ ~ both the "infected' ' and "all' ' family groupings were found to be not significantly different from a normal distribution (a < 0.05). The Poisson distribution was not an ade- quate model of the distribution of index values among the resistant families (a < 0.05) and this distribution demonstrated a sigruficant (a < 0.01) right skew. The distribution was found to fit the negative binomial d i s t r ibu t i~n~~ (a < 0.05) by both the chi-square and T-statistic goodness-of- fit tests. This demonstrates that the mean index value of the resistant families was shifted toward the primitive end of the scale. The mean of the index value of the infected families (57.5) was significantly greater than the mean of all families (5 1.2) (a < 0 .O5) while the 95 % confidence in- tervals of the mean of the resistant families (53.4) overlapped those of both of the other groups.

This analysis would seem to suggest con- currence with the assessment of De Cleene and De Ley1' that both primitive and advanced families are relatively resistant to infection. How- ever, the index values of the infected families range from 37 to 83 while those for the resistant families ranged from 38 to 87, giving no clear indication of the validity of judging either more primitive or more advanced families as being likely to be more or less susceptible to A. rhizogenes infection. The three resistant dicotyledonous families, Cactaceae, Gesneriaceae, and Lamiaceae, have index values of 53, 70, and 72, respectively, while the three strongly susceptible families, Fabaceae, Rosaceae, and So- lanaceae, have values of 48, 43, and 68, respectively. It is not yet completely clear whether these analyses reveal actual patterns or whether they still suffer from insufficient and biased sampling. Although not specifically analyzed, it would be relatively easy to show that infection trials to date have been biased toward north temperate species. S p ~ r n e ' + ~ also discussed this as a factor which limits taxonomic pattern analysis in plant families in general.

D. Relation to the infection Process

A critical question in an analysis of this sort

is whether infection with a particular bacterium can be used as a taxonomic character. In order to answer this question fully, it should be possible to identify the factors which allow that infection. So far, that is not possible, as has been shown above. However, there are a number of characters which can be identified, at least in principle, to occur if successful infection occurs. An examination of the infection process demonstrates a number of steps which must occur before the appearance of infection symptoms. (1) Early on is the production of substances by the plant which indicate wounding and receptivity to the Agro- bacterium. These have been discussed above, but it is not yet clear whether any of several such compounds can lead to bacterial induction and chemotaxis or only a few, specific molecules are required. (2) Chemotaxis of the bacterial cell toward the plant cell results in (3) recognition by the two cells based on cell surface receptors. Again, these are poorly understood. Although it is also unclear whether this is a requisite, (4) binding of the bacterium occurs and this can be blocked by competitive bacteria or an abundance of pectic s ~ b s t a n c e s . ~ ~ Binding is followed by (5) bacterial gene activation and (6) transfer of the Ri genes to the plant genome. These must be (7) incorporated into the plant genome and then (8) recognized as a valid part of the genornic information for transcription, a sequence which could be referred to as genome compatibility. (9) The translation of the Ri genes then must result in a chemical environment in the cell which leads to (10) the production of the characteristic symptoms. This last step is clearly missing in many of the monocots in which gene incorporation and opine synthesis have been shown biochemically. Rather simply, the occurrence of a successful infection clearly demonstrates the presence of all of the critical characters in the host plant; lack of infection says at least one is missing, but, unless a gene probe shows DNA incorporation, it is not clear which or how many of the requisites are missing. It remains for further investigation to determine whether these characters can be applied more broadly to plant-bacterial interactions and whether any or all might still have application in systematic studies.

A. Description of acterial Strains

1. Natural Strains

A number of natural, wild-type strains or selected derivatives from these have been used for the induction of hairy roots. Many of the earlier studies referred to the strain employed by a number only identifiable within the laboratory in which the studies were done and are not easily identifiable as being related to, or even the origin of, strains available in a systematized collection; these are here referred to as wild-type strains rather than unknown in Table 1. The latter designation is used when no strain is designated and the specific strain cannot be inferred through the description or through direct reference to other work. In general, the wild-type isolates are not sufficiently characterized to determine a strain, or even a biovar, designation. An exception is the report of Munnecke et al.@ This group studied hairy root of roses and inoculations of the isolates on tomato, tobacco, and roses. Based on the reported acid litmus milk reaction, all 18 isolates can be tentatively assigned to biovar 2. This is also true of the earlier work of Riker et al.66q67

The abbreviations used in the following dis- cussion are ATCC = American Type Culture Collection; ICPB = International Collection of Phytopathogenic Bacteria, Berkeley, CA; NCPPB = National Collection of Plant Pathogenic Bac- teria, Harpenden, U.K.; PC = Phabagen Col- lection, The Netherlands.

A strain which has been used widely, but is generally only moderately infective, is ATCC 1 1325. This was originally isolated from apple hairy root around 1942.68 This strain is biovar 2 and produces nopaline as its major opine; this latter has caused some to consider this strain to be a close relative of A. turnefaciens or even a strain of this latter species.

A number of other strains have been used which are part of one or another organized collection. ATCC 15834 ( = NCPPB 2629), NCPPB 1855 ( = LBA 9402), PB 27 18, and A4 ( = ATCC 31798) are biovar 2 strains which produce agro-

pine and mannopine as the opines, making them agropine-type strains. A4 was originally isolated by Dr. Peter Ark at the University of California- Berkeley from naturally infected roses. 69 NCPPB 8 196 ( = LBA 9403) and ICPB TR7 ( = NCPPB 2626, ATCC 25818) are also biovar 2 strains but which lack the ability to synthesize agropine, causing mannopine to be the major opine (mannopine-type s trains). ICPB TR I07 ( = NCPPB 2628), NCPPB 2655,2657, and 2659 are biovar 1 type strains with cucumopine as the major opine. Costantino et aL70 have reported that these last three strains all harbor the same Ri plasmid and so are referred to here collectively as 2655 (Table I). ICPB TR105 is a biovar 2 strain of the agropine type.71 Although this strain has been reported to be identical to strain K47, the infection of four gymnosperms was substantially different when the two strains were compared;40 however, both strains were capable of infection in all four cases. Strain IS47 and strain K49 are isolates from infected Prunus,'+O while the origin of TR105 is unknown. Both TRIO5 and K47 are agropine strains, while the opines of strains K49 (unknown biovar type), ATCC 13332, and 13333 (biovar 1 strains) have not been reported. Strain K599 is apparently an isolate from A ~ s t r a l i a ~ ~ which produces cucumopine; no other information is available on this strain. At present, there does not appear to be a relationship between the major opine produced by a strain and its infectious potential, although most of the more infectious strains are agropine strains. Since the role of the opine, if any, in the infection and transformation processes has not yet been adequately determined, it is unclear whether this correlation of opine type and infectivity is causal or circumstantial .

Strain 232 (= ATCC 39207) is a spontaneous mutant derivative of TR105 which was selected for greater rooting capacity on carrot and parsnip.32 This strain is being studied for its potential to increase the root volume of nursery stock of almond32 and olivee7' Before planting, the population of virulent A. rhizogenes is re- duced by inoculation with the agrocin-producing A. r a d i o b a ~ t e r . ~ ~ ? ~ ~ This strain has also recently been used for the transformation of alfalfa.76

Several strains were excluded from Table I because only a few infection trials have been

reported. PB 271877 and NCPPB 265778 have only been reported to infect carrot. Additional strains (ICPB TR102, A2, and A47) have only appeared in the literature in infection trials on Papaver s~mni f e rum~~ and, in the case of the latter two strains, Catharanthus roseuss0 on which they were infectious; no other information about these strains has appeared. Strain IS49 has only been tested on A bies procera, Pseudotsuga menziesii, and Tsuga heterophylla, on which it was infectious, and Pinus ponderosa, on which it was not. A recently described wild strain is Ar M- 123, isolated from hairy root of muskmelon.81 This strain is mannopine positive but agropine negative. The biovar characterization has not yet been published, but it has been used in attempts to infect muskmelon (Cucumis melo) and ornamental kale (Brassica oleracea L. var acephala cv Shirohato), on which it was infectious, as well as Pelargonium sp., Sinningia speciosa Benth. and Hook., and ornamental tobacco (Nicotiana sp.), on which it was not. Benjarna and DaoudU2 isolated 44 pathogenic and nonpathogenic Agro- bacterium strains from root tumors of almond, peach, and plum in Morocco. Of 14 biovar 2 strains from this collection tested for infection of Kalanchoe tubiflora, all but 3 produced roots at the site of infection, and so could presumably be classified as Agrobacterium rhizogenes. How- ever, the opine isolated from infected tissue was nopaline, characteristic of A. rhizogenes ATCC 1 1 3 25 and A. tumefaciens strains.

2. Engineered Strains

In addition to natural or selected strains of A. rhizogenes, a number of strains have been engineered to be more infective or to carry more easily detected markers with which to confirm transformation. These strains are either A. rhizogenes into which have been inserted, by con- jugation, A. tumefaciens plasmids to increase the host range (or as a binary vector for the insertion of a selectable marker) or the reciprocal, in which an A. rhizogenes plasmid has been inserted into A. tumefaciens to confer the rooting characteristic to the infection. In addition, various other plasmids , generally from Escherichia coli, carrying one or more marker genes, may also be incor-

porated into the Agrobacterium strain being used, or one or more genes from such plasmids may be inserted into an Agrobacterium plasmid.

The strain 1855 ( ~ G A 4 9 2 ) ~ ~ is A. rhizogenes strain NCPPB 1855 into which is inserted the A. tumefaciens plasmid pGA492. This is a binary vector carrying the selectable marker for a fusion protein of nopaline synthase and the neomycin phosphotransferase gene (nptII) from the bacterial transposon Tn5, conferring kanamycin resistance. This strain has only been used so far to infect Stylosanthes humilis cv Paterson and is included in Table 1 under 1855. This species has also been successfully infected by NCPPB 1855 without the binary vector. A rather interesting set of experimentss4 demonstrated that A. rhizogenes 1855 exhibited greater rooting potential when co- incoulated with A. tumefaciens strains GV3850 (disarmed derivative of C58C lRS) or LBA4.404. Rooting was particularly stimulated when the A. tumefaciens strain contained an E, coli pUCD 1 186 plasmid containing pTiC58 with an active trans- zeatin gene.

A second group of engineered strains with A. rhizogenes parentage is based on the strain

4 4 . One such strain is A41pARC8 ,85 This is A. rhizogenes strain A4 into which has been inserted a Ti plasmid containing, as the selectable marker, the Tn5 npt structural gene flanked by the nopaline synthase promoter and terminal sequence. There is also a tetracycline resistance marker on the vector plasmid. A similar strain is A4/ P A R C ~ , ~ designated by them as strain 1072. This differs from the forrner in that the nopaline synthase gene is intact (conferring the ability to synthesize nopaline in addition to mannopine and agropine) and th re%X&npt.gene present. These strains are refe 4 ed to collectively in Table 1 as A4/ARCx. 1

The group of wains described by Lam et al? (designated BL3- in-~sists completely of derivatives of A, rhizogenes strain TR 105. The Ri plasmid has had Tn5 inserted which confers resistance' to kanarnycin and neomycin, by con- jugation with E. coli strain 1830 as donor. The major strain used in this group has been BL3 11 which is infective on several s p e ~ i e s . ~ ~ * ~ ~ Others which have been used in attempts at rhizogenesis are spontaneous mutants of this basic type and include BL3 17 and BL322. Both strains are at

least somewhat positive in inducing root initiation on red beet, and BL322 induced shoot formation on parsnip.86 Strain BL3 11 has also been used to transform Rhizobium trifolii and R. mel- iZ~ t i . *~ Except in the case of one spontaneous mutant strain, the transformed R. trifolii was incapable of nodulation, but it did initiate root formation on the same host plants as have been tested for strain BLd 11. While the nodf mutant appeared to be highly infective, it was not very effective in fixing nitrogen. The transformed R. meliloti was capable of nodulation at a greater frequency than the normal strain and did not induce root A similar strain, LPR5079, has been described. 8g LPR.5079 is R. trifolii which carries pRi 1855 and is infective on carrot.

A very widely used engineered group of strains are those based on A. tumefaciens strain C58C 1 into which A. rhizogenes plasmid pRiA4 has been inserted, here collectively referred to as C58C 1 a. These have been variously designated as A4T, C58C1 (pRiA4b), R1000, A4TII1, or R 1500. The first three are essentially equivalent, and, in fact, the second is derived from the first. Strain A4TIII is strain A4T containing an engineered A4 plasmid which carries the intermediate vector, pAMNeolO. This insert contains a car- benicillin resistance gene plus the chimeric nopaline s ynthase-npt gene Strain R 1500 is the RlOOO strain into which has been inserted the nptII gene inserted into the Hind III fragment 21 of the Ri p l a~ rn id .~~ The nptII gene confers kanamycin resistance. Two more strains have been engineered in this group, R16OO and R1601 ;92

these have an additional insert of the E. coli pTVK291 cosmid containing the vir region of the A. tumefaciens " supervirulence" plasmid, pTiBo542, inserted into either RlOOO or R1500, respectively. Four additional strains lacking rolA (Rl5Ol), roZB ( R I ~ o ~ ) , rolC (R l5O3), and rolD (R1504), loci on the T,-DNA which control infection phenotype, have also been constructed and found to be infective on Kalanchoe diagremontiana and Nicotiana tabacum var Xanthi.91 These constructions have also been designated R1022 (rolA-), R1023 (roZB-), R1016 (rolC-), R124.4 (roZD-), and R1020 (no rol locus assigned)30 which were also found to be infectious

on Kalanchoe diagremontiana. Boulton et a1. 35 have inserted the maize streak virus into this, and other, backgrounds as a detectable marker to test agroinfection of various grass species. Other viral genornes have also been used for different hoskg3 Several strains have been designed to investigate the role of the T,-DNA in root transf~rmation.~~ These consist of the A4T background with several additional plasmid fragments which disarm or complement the TL-DNA of the A4 plasmid. Two strains used extensively by these authors are BNlOlO and BN1010::pEM15. Although all of these A4-derived strains are grouped together here as C58Cla, it should be noted that differences in infectivity in various species have been n ~ t e d . ~ ~ . ~ ~

A similar strain is based on A, tumefaciens strain GV3 1 0 1 . A. tumefaciens strain CV3 10 1 is a rifampicin and gentamicin resistant derivative of strain C58;95 for that reason, this strain is included under C5 8C 1 a.

Another strain based on C58C1 (designated C58Clb) is that described by Hansen et aleg6 In this case, the A . tumefaciens strain carries pRi15834. This strain has been used to transform Lotus corniculatus L. cv Rodeo in order to produce transgenic root nodules following coinfec- tion with Rhizobium loti. In strain GV3101 (pRi15834), the Ti plasmid of A. tumefaciens strain GV3 101 is replaced by the Ri plasmid from A. rhizogenes ATCC 15834. This engineered strain has been used in the unsuccessful attempt to transform five cultivars of pea.97 Other A. tumefaciens strains were also unsuccessful in this trial, which supports the work of Bercetche et al.98 and Hobbs et a1 .99 Considerable strain var- iation exists with both A. tumefaciens and A. rhizogenes for infection of this crop plant. For the same reasons as stated for GV3101 (pRiA4) above, this strain is included under C58C 1 b.

Strain C58C 1 c in Table 1 is A. tumefaciens strain C58C 1 which contains pRi8 196?'O A strain incorporating pRi1855 is LBA1334, '01 which is a derivative of A. tumefaciens strain C58C9. This is not included in Table 1 because it has, so far, only been used for the transformation of Trifol- ium repens. Strain C58Cld is, again, the C58 strain in which has been placed the Ri plasmid of A. rhizogenes TR 105 .40

elative lnfection of the Tested Strains

Among the strains listed in Table 1, it can be determined which have infected the greater number of species, and so, may be more infectious than other strains. In this analysis, it was decided to not include the strains discussed above which were not included in the table because the results of a single or just a few trials could easily and seriously over- or underestimate the true infectivity of the strain.

The strains included in Table 1 have been tried on a minimum of 4 taxa (C58Cld (pRi- TR 105)) to a maximum of 2 16 trials (TR7) (Table 2). The very large number of trials for this last strain is due, in large part, to the work of De Cleene and De Ley.1° A much more diverse history is found for strain A4, which has undergone 169 trials. To calculate the percentage infection of these strains, the number of positive infections was divided by the total number of trials; where equivocal results have been obtained ( rt: in Table l ) , 0.5 was added to the positive results per in- cident. Two categories not evaluated were the unknowns (109 trials) and wild isolates (17 trials). It was not felt that further evaluation of these groups would yield any useful information since there is probably considerable overlap between both of these groups and the named isolates.

Based on the percentage of successful infections (Table 2), the natural strains can be placed in descending order as follows: K47 = K599 = HRI > TR105 > 15834 > A4 > 1855 > 2655 > TRlOl > 8196 > 232 > 11325 > 43057 > TR7 > TR107 = 13332 = 13333. It should be noted that K47, 232, TR107,2655,43057, U 9 9 , and HRI have been inoculated in infection trials a relatively few number of times (5 10 for each), and so these results are tentative. It is clear that 1855, 15834, and A4 are highly infectious, ex- hibiting infection percentages of 9 1 .9, 95.5, and 9 1.7 with 86, 56, and 169 trials, respectively. Strain TR105, at 97%, has been tried 33 times. Strains 13332 and 13333 have each undergone 1 3 trials, but have failed to infect any plant tried. Although such data are not usually available from the literature, Savka et al.72 reported the frequency of root production compared to the number of inoculations. Strain K599, which was in-

TABLE 2 Success of Infection of A. rhizogenes Natural and Engineered Strains

Strain

Unknown Wild type Natural

A4 K47 ICPB TR7 ICPB TRIO1 ICPB TRIO5 232 ICPB TRIO7 NCPPB 1855 NCPPB 2655 NCPPB 8196 ATCC 1 1325 ATCC 13332 ATCC 13333 ATCC 15834 ATCC 43057 K599 HRI

Engineered BL3 - C58C1 a C58Cl b C58Cl c C58Cl d A4/ARCx

No. of

trials Infected

("w

Note: Abbreviations of the strains are as described in the text.

fectious on all 10 soybean cultivars tested, produced roots in 74 of 200 inoculations (37%). Strain 8196, 1855 and A4, also inoculated at 200 sites, were infectious 7, 3.5, and 7 % of the time, respectively.

Among the engineered strains, a similarly broad range of infection capability was found following the descending order of A4/ARCx > C58Cla > C58Clb > C58Cld > BL3- >> C58Clc. Only strains C58Cla and C58Clc have undergone an appreciable number of trials, 77 and 36, respectively; the others have been tried 13 or fewer times. It is important to note that the strain which has been least manipulated from the parental type, A~IARCX, exhibited the highest

infection percentage, 100%. This is also the only one which significantly exceeds the infection percentage of its A. rhizogenes parent or donor strain. The infection of C58Cla is comparable, at 93.8%, to the plasmid donor strain, A4, at 91.7%. How- ever, it should also be realized that C58Cla con- sists of a variety of individual strains, which have also exhibited differences in their ability to infect a particular host. Except for strain C S C kc and the BL3 - group, the other engineered strains have infection percentages near, but lower than, the donor strains. The BL3 - group had an infection percentage of 44.4% with a significant number of equivocal results compared to its parent strain, TRlO5, with a percentage infection of 97%. It would appear that the BL3 - group has been substantially disabled by its Tn5 insert. Strain C58Clc, with 11.1% infection, stands in stark contrast to its donor strain, 8196, with 74.5% infection.

A major reason for engineering a strain is to confer some property of the parent or donor strain not found in the native strains. In some cases, the addition of a selectable marker is the goal of the manipulation. In the case of the C58C1 strains, it is often hoped that the rooting characteristic will be provided by the Ri plasmid, while the supposedly greater infection of the A. tumefaciens will extend the range of plants which can be induced to root. However, in some cases, the engineered strain has infection characteristics much different from either the host or donor strains. Infection by engineered strains differed from native strains in four gymnosperms. 40 A. tumefaciens C5 8 infected A bies procera at 29%, Pinus ponderosa at 9%, Pseu- dotsuga menziesii at 25%, and Tsuga heterophylla at 7%. Infection by C58C 1 a (pRiA4) was 5, 0, 15, and 0%, respectively, while that of the plasmid donor strain (A4) was 90, 35, 30, and 80%, respectively. C58C 1 c (pRi8 196) was not infectious on any of these species; the donor strain (8196) was not tested. C58Cld (pRi- TR105) was infectious in 15, 0, 5, and 5% of the trials, and the donor strain (TR 105) yielded infection in 60, 75, 40, and 40%, respectively. In Robinia pseudoacacia, the A4 strain is also infectious, comparably so to various infectious A. turnefaciens strains,lo2 but the engineered strain RlOOO (C58Cla) is not. The authors sug-

gest that an insufficient sample size may be responsible for the negative results, but infectious strains showed infectivity with fewer trials; the parent strain C58 was not tested. More careful examination of the engineered strains listed in Table 1 shows that in no case is the engineered strain more infectious than its donor strain, and in several cases is less so. In the reciprocal crosses, in which a Ti plasrnid is inserted into an A. rhizogenes parent (A4/pARCx), the infection of the engineered strain is the same as that of the parent strain, except for Tagetes patula, in which the A4 parent strain has not been tested. The engineered BL3 - strains, derived from TRIOS, match the infection of the parent strain in some cases, give mixed results in others in which the parent strain infects, and is noninfec- tious in two species tested, Daucus carota var sativa and Dioscorea alata, both of which are infected by the parent strain. It would appear from these results that there are a number of instances in which the infection is regulated by the parent strain and, in others, the engineered strain infection is less than that of either the donor or the parent strain. There must, therefore, be an assessment of strain specificity before engineered strains are deemed to be required or useful. The major exception would be for the insertion of a selectable marker, although it would appear that additional work is required to de- velop engineered strains which are not significantly altered in their infectivity.

V. CONCLUSIONS

The results presented here require a major revision of the generally held views that A. rhizogenes is generally, if not always, less infective than A. turnefaciens and that the engineered strains of both these bacteria possess superior infection capabilities. In a number of instances, A. rhizogenes is as effective, or more so, in transformation of the host plant compared to A. turnefaciens. It would also appear that, except in some isolated cases, that the primary advantage that an engineered strain possesses is the presence of a selectable or detectable marker. This may change with further work, but the only strains which presently deserve the descriptor, " superinfec-

tive", are HRI, K47, K599, and the A41ARCx group. Again, it should be cautioned that all of these strains have been used very infrequently in infection trials.

The occurrence of infection by A. rhizogenes could not be definitely correlated with any of 19 characters for which an appreciable amount of data could be found. Four families, the Cacta- ceae , Gesneriaceae , Lamiaceae , and Liliaceae, were found to be almost universally resistant to infection, with some minor, but nevertheless interesting, possible exceptions noted in the latter two families. Separation of these families from the major infected families, the Rosaceae , Fa- baceae, and Crassulaceae, could be obtained based on plant phenolics, but were not separated from the Solanaceae and Asteraceae .

Two families were identified which will probably continue to remain resistant to infection, the Cactaceae and the Gesneriaceae. Both have had several species inoculated with multiple strains of bacteria. De Cleene and De Ley6' did report the infection of members of these families by A. tumefaciens, suggesting that there may yet be quantitative differences between these two species. The other two families which have been consistently resistant to infection, the Lamiaceae and Liliaceae, are more enigmatic. Wound re- sponses sirnilar to those reported for Asparagus spp. and Narcissus spp. when inoculated with A. tumefaciens have been found in this laboratory on Aloe variegata (Liliaceae). Further examination by opine analysis or DNA probes may reveal the presence of transformation. In the Lamiaceae, only Coleus blumei has been reported as successfully infected. This again contrasts to the experience with A. tumefaciens in this fam- i l ~ , ~ ' but many fewer species have been inoculated with the "hairy root" organism. Consid- ering the abundance of secondary metabolites in the mints and related plants, it would be valuable to find or select a strain which was capable of consistent infection. It is interesting to note that Coleus is in the most advanced section of the family. This suggests that the particular chemical constituents of the other sections may be inhibitory to bacterial infection, that there is a fairly simple genetic barrier to infection since it does not appear in the more primitive members but recurs in the advanced section, or that Coleus is

improperly placed within the Larniaceae, as has been suggested previously. The Verbenaceae , the family to which this section has sometimes been assigned, has also resisted infection.

ACKNOWLEDGMENTS

I thank Chris Gussman for his skillful exe- cution of some of the inoculations and our many useful discussions. I also thank the Elsa U. Par- dee Foundation for partial support of this work, Hector E. Flores, Pennsylvania State University, for providing me with one of the bacterial strains, and Robert A. Smith for his review of the manuscript.

REFERENCES

1. Chilton, M.-D., Tepfer, D. A., Petit, A,, David, C., and Casse-Delbart, F., Agrobacterium rhizogenes inserts T-DNA into the genornes of the host plant root cells, Nature, 295, 432, 1982.

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https://www.researchgate.net/publication/7185727_Hairy_roots_are_more_sesitive_to_auxin_than_normal_roots?el=1_x_8&enrichId=rgreq-087fbcfb2821d3ba694426a1e8c2921b-XXX&enrichSource=Y292ZXJQYWdlOzI0ODkzNjIwOTtBUzoxMzkxNDYzMzcxOTgwODBAMTQxMDE4NjQ4MDY2MQ==



https://www.researchgate.net/publication/258348113_Thiophene_accumulation_in_relation_to_morphology_in_roots_of_Tagetes_patula_-_Effects_of_auxin_and_transformation_by_Agrobacterium?el=1_x_8&enrichId=rgreq-087fbcfb2821d3ba694426a1e8c2921b-XXX&enrichSource=Y292ZXJQYWdlOzI0ODkzNjIwOTtBUzoxMzkxNDYzMzcxOTgwODBAMTQxMDE4NjQ4MDY2MQ==





https://www.researchgate.net/publication/257040344_Effects_of_Gibberellic_Acid_on_Hairy_Root_Growth_in_Datura_innoxia?el=1_x_8&enrichId=rgreq-087fbcfb2821d3ba694426a1e8c2921b-XXX&enrichSource=Y292ZXJQYWdlOzI0ODkzNjIwOTtBUzoxMzkxNDYzMzcxOTgwODBAMTQxMDE4NjQ4MDY2MQ==



https://www.researchgate.net/publication/246996082_Upstream_non-coding_region_which_confers_polar_expression_to_Ri_plasmid_root_inducing_gene_rol_B?el=1_x_8&enrichId=rgreq-087fbcfb2821d3ba694426a1e8c2921b-XXX&enrichSource=Y292ZXJQYWdlOzI0ODkzNjIwOTtBUzoxMzkxNDYzMzcxOTgwODBAMTQxMDE4NjQ4MDY2MQ==




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https://www.researchgate.net/publication/20663800_Transformation_of_plant_cells_via_Agrobacterium?el=1_x_8&enrichId=rgreq-087fbcfb2821d3ba694426a1e8c2921b-XXX&enrichSource=Y292ZXJQYWdlOzI0ODkzNjIwOTtBUzoxMzkxNDYzMzcxOTgwODBAMTQxMDE4NjQ4MDY2MQ==

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Host range and implications of plant infection by Agrobacterium rhizogenes

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