SEASONAL FLUCTUATIONS AND SPATIAL DISTRIBUTION OF THE MAJOR PHYTONEMATODES INFESTING A VINEYARD AT...

Zagazig J. Agric. Res., Vol. 42 No. (1) 2015

SEASONAL FLUCTUATIONS AND SPATIAL DISTRIBUTION OF THEMAJOR PHYTONEMATODES INFESTING A VINEYARD AT SHARKIAGOVERNORATE, EGYPT

Amr M. El-Marzoky*, A.A. Salem, M.E. Mahrous and Mervat H. IbrahimPlant Prot. Dept., Fac. Agric., Zagazig Univ., Egypt

ABSTRACT

Seasonal population dynamics and spatial distribution of the predominant phytonematodes infesting a vineyard in Belbies district,Sharkia Governorate, Egypt were studied. Soil population density of the four most important plant-parasitic nematodes were monitored on King Ruby cultivar over 12 months period started from December 2011 to November 2012. Numbers of Rotylenchulus reniformis followed by Meloidogyne incognita were markedly higher as compared to those of Tylenchulus semipenetrans and Pratylenchus spp. at all months. The maximum densities of these nematodes were detected in spring (May) with levels of 360, 142.80, 50 and 45 individuals; whereas, their lowest numbers were determined in summer (August) with population density of 170, 33, 15 and 22 individuals per 250 g soil, respectively. Horizontal and vertical distribution of the common phytonematodes were studied on Flame cultivar at the same vineyard. Nematode densities were estimated at five different horizontal positions far from the vines including in rows (0-50 and 51-100 cm) and inter-rows(0-50, 51-100 and 101-150 cm). Moreover, numbers of nematodes were detected at 0-20 and 21-40 cm depth in the horizon 0-50 cm far from vine trunk. It was clear that, total numbers of all encountered nematodes were significantly differed (P≤0.05) from distance to another. The highest total numbers of nematodes were found at distance of 51-100 cm between rows, followed descendingly by 0-50 cmin rows, while moderate value was found in the distance 0-50 cm between rows ; however, the lowest numbers were detected in distances of 51-100 cm in rows and 101-150 cm between rows . Percentdistribution at these distances were 37.06, 26.58, 16.51, 11.23 and 8.62%, respectively. On the other hand, around 62% of total nematodes were occurred at the top 20 cm compared to about 38% found

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El-Marzoky, et al.

at the deeper depth 21-40 cm. This finding is true for M. incognita, R. reniformis, T. semipenetrans and Helicotylenchus spp. Contrarily, individuals of Xiphinema spp. were detected with higher numbers in deeper stratum as compared to lower values in the top one. Based on these data , sampling of grapevines at distance of 51-100 cm from tree trunk between rows to a depth of about 20 cm in spring season is considered the best option to determine population density of phytonematodes infecting grapes. There is notable exception to this generalization , since Xiphinema spp. prefer deeper soil layers and tend to increase depth of sampling more than 20 cm. Key words: Plant parasitic nematodes, grape, seasonal fluctuations,

horizontal and vertical distribution.

INTRODUCTIONGrape (Vitis vinifera L.) is

considered the first deciduousfruit crop in the world and thesecond major fruit crop in Egyptafter citrus. Egypt occupies animportant position inviticulture of the world andranks the 13th place in the grapeproduction .The total areacultivated with grape in Egyptreached 66262 hectares(163736.1faddans) producingabout 1378815 tons (FAO, 2012).The flavinoids present in grapeact as antioxidants and reducethe damage caused by fullradicals. Due to its medicinalproperties, grape juice ispopularly known as "nectar ofgods". It is easily assimilatedand is a good remedy forconstipation, rheumatism, skinand liver disorders (Varmudy,2011).

Plant parasitic nematodes arecommonly found in vineyards inEgypt as well as in all regionsof the world and are oftenassociated with reduced vinevigor. An annual yield loss of12.5% in the total grape hadbeen determined by Sasser and

Freckman (1987). Root-knotnematodes, Meloidogyne spp. have acosmopolitan distribution andare a major productionconstraint especially in sandysoils. Moreover, lesionnematodes, Pratylenchus spp.; thereniform nematode, R. reniformis andthe citrus nematode, T.semipenetrans have been reported tocause considerable damage tograpevines and commonly found invineyards (Raski et al., 1973;Ferris and McKenry, 1975;Loubser and Meyer, 1987;Pinkerton et al., 1999; Belair et al.,2001; Kesba, 2003; Aballay et al.,2009; Ibrahim and Mokbel, 2009).

Information of the horizontal,vertical and seasonaldistribution of the major plantparasitic nematode genera isvery important to select theappropriate sampling proceduresfor nematode detection andquantification .These data alsohelp to identify factors thataffect nematode dynamics (Quaderet al., 2003). A betterunderstanding of the spatial

* Corresponding author: Tel. : +201228536236

E-mail address: [email protected]


distribution of individuals bothhorizontally and vertically isessential to accurately buildsampling strategies andexperimental designs (Noe andCampbell, 1985; Villate et al.,2008). Moreover, thisinformation is very necessaryfor understanding ecosystembiology (Norton, 1978; Bird andRamsdell, 1985).

The vertical distribution ofplant parasitic nematodes insoil may be affected by rootdistribution, height of watertable, soil moisture,temperature, soil texture andrainfall (Potter, 1967; Miller,1972; O’Bannon et al., 1972;Brodie, 1976). There is someevidence that verticaldistribution patterns could beinfluenced by season (Norton etal., 1971; Ferris and McKenry,1975). Wallace (1963) indicatedthat root distribution is themain factor in the verticaldistribution of plant-parasiticnematodes and other physicalfactors play an importantsecondary role. Generally, dataconcerning seasonal fluctuationand spatial distribution ofplant parasitic nematodes in therhizosphere of grapevines grownin sandy soil of SharkiaGovernorate are still lacking.

MATERIALS AND METHODS

Experimental VineyardA series of experiments were

conducted in established 20-

years old, own rooted vineyard located in Sekem Farms, Belbies district, Sharkia Governorate, during the period from December 2011 to November 2012. This vineyard was about three faddans, planted with Flame seedless and King Ruby cultivars. The vine spaces were approximately 3 m between the rows and 2 m within the rows. Vines in the farm were drip irrigated.

Soil subsamples were taken from the vineyard to the depth of 25-35 cm and mixed to form composite sample which was processed for mechanical analysis in the Central Laboratory, Faculty of Agriculture, Zagazig University.The soil texture was classified as sandy loam with mechanical analysis of 15.75% clay, 12.45% silt and 71.80 % sand.

Initially, 20 soil samples were randomly collected from thevineyard and assessed for nematode extraction to determinepopulation density to locate plots with heavy infestation of plant parasitic nematodes for further studies. Each plot consisted of 4 rows x 6 vines. One composite soil sample was collected from ten trees in eachplot. Vines in the highest five plots containing phytonematodes were marked for future sampling.These data were not included in this study.

El-Marzoky, et al.

Sampling, Extraction, Identification and Counting of Nematodes

A handful of soil containing feeder roots were collected one day after irrigation using a shovel. Five subsamples were mixed together to form a composite sample. Samples were placed in polyethylene bags and transferred directly into laboratory and kept in a refrigerator at temperature of 6-7 ˚C until they were processedfor nematode extraction in the next day. After appropriate mixing of each soil sample, 250 g of soil were assessed for nematode extraction. Galled roots were collected and stored in formalin 4%. Nematodes were extracted using a combination ofsieving and Baermann trial technique (Hooper et al., 2005).

Nematode identification was based on morphology of adult andlarval forms according to Mai and Lyon (1975) and Siddiqi (1986). Nematodes were counted using Howksely counting slide under a research microscope (100x magnification) and identified to generic level. To determine species of root-knot nematodes infecting grape, about 20 adult females were isolated from galled roots. Species of Meloidogyne were identified on thebasis of perineal pattern according to Taylor and Sasser (1978) and Eisenback et al. (1981).The perineal patterns were prepared as described by Taylor and Netscher (1974). Seasonal Fluctuations of FourImportant Plant Parasitic

Nematodes Infecting King RubyGrapevine Cultivar

Population density of four important plant-parasitic nematodes i.e.; M. incognita, R. reniformis, T. semipenetrans and Pratylenchus spp. was detected in the vineyard at monthly intervals during one year started from December 2011 to November 2012 on King Ruby cultivar. Five soil subsamples containing feeding rootlets wererandomly collected from five trees in each plot as previouslymentioned. Samples were taken ata depth of about 25-35 cm at distance of 0-50 cm far from tree trunk. Data concerning air temperature were obtained from Abou-Kapper Meteorological Station. Spatial Distribution of the Major Phytonematodes Infecting Flame Grapevine Cultivar

This trial was conducted during February 2013. To study horizontal distribution, five trees were chosen in each of thefive plots previously determined. Sampling positions (S1- S5) for horizontal distribution between rows and vines are illustrated in Fig. 1.Samples between rows (S1 - S2 - S3) were collected at three distances 0-50 , 51-100 and 101-150 cm far from vine trunk, while those between vines (S4 andS5) were obtained at two distances 0-50 and 51-100 cm away from vine trunk. All samples were taken at the depth


of 25-35 cm. Subsequent samples were not taken from the marked trees to avoid errors introduced

by root-damage during previous sampling.

Fig. 1. Schematic diagram showing sampling positions (S1 – S5) relative to the grapevines and row spacing in Sekem Farm vineyard; S1, S2 and S3 in the distances of 0-50 , 51-100 and 101-150 cm far from vine trunk (between rows) and S4 and S5 in the distances of 0-50 and 51-100 cm far from vine trunk (between vines)

The vertical distribution was designed by the same manner in another five trees chosen in each plot at depths of 0-20 and 21-40 cm at the distance of 0-50cm away from vine trunk between vines. It is necessary to mention here that sampling direction was changed around vines in different replicates. The abundance percentage of eachnematode species present at eachdistance or depth was calculatedaccording to the equation applied by McSorley and Dickson (1990) as follows:

Data were subjected to statistical analysis using F test. Means were compared by Duncan's multiple range test at 5% level of probability according to Snedecor (1966).

RESULTS AND DISCUSSION

Seven genera of true plant parasitic nematodes were found in association with roots of King Ruby and Flame seedless grapevine cultivars in the study

Between

vines

Betweenrows

50 cm 100 cm 150 cm

El-Marzoky, et al.

area in Belbies district, Sharkia Governorate. These genera were root-knot nematode, Meloidogyne (Goeldi); reniform nematode, Rotylenchulus (Linford and Oliveira); citrus nematode, Tylenchulus (Cobb); lesion nematode, Pratylenchus (Filipjev); spiral nematode, Helicotylenchus (Steiner); stunt nematode, Tylenchorhynchus (Cobb) and dagger nematode, Xiphinema (Cobb). Examination of perineal pattern of adult Meloidogyne females revealed the presence of chieflyM. incognita (Kofoid and White) Chitwood.Seasonal Fluctuations of FourImportant Phytonematodes infecting King Ruby GrapevineCultivar

Soil population density of theendoparasites M. incognita and Pratylenchus spp. as well as the semiendoparasites, R. reniformis andT. semipenetrans was estimated at monthly intervals started from December 2011 to November 2012. Data in Fig. 2 indicated that inthe term of prevalence numbers of M. incognita and R. reniformis were relatively higher than those of T. semipenetrans and Pratylenchus spp. It was evident at all examination dates, since generalmeans of the above mentioned nematodes were 62.70, 239.07, 30.50 and 29.75 individuals per 250 g soil, respectively. On theother hand, seasonal population behavior of these nematodes showed about the same trend

throughout the whole year studied. However, infestation ofgrapevines with each of these nematodes was found with different population density throughout the year seasons. Onepeak or suitable period for development and reproduction occurred in spring (May). In another words, population density of the tested nematode species reached their maximum levels of infestation in spring (May) with values of 142.80, 360.00, 50.00 and 45.00 individuals per 250 g soil, respectively. On the contrary, numbers of these nematodes were found in their minimum levels insummer (August) with population density of 33, 170, 15 and 22 individuals per 250 g soil for the above mentioned nematode species, successively. However, soil population density of the tested nematodes were less or more similar in winter (February) and autumn (November)seasons with values of 50 and 76juveniles for M. incognita, 200 and198 juveniles or preadult females for R. reniformis, 20 and 32juveniles for T. semipenetrans and 30 and 25 individuals for Pratylenchus spp., consecutively.

These results are in harmony with those reported by Kesba (2003) who indicated that Meloidogyne and Rotylenchulus were highly destructive to the most grape cultivars which consideredas poor hosts for the citrus nematode T. semipenetrans. On the


other hand, Riad (1974) studied population behavior of M. incognita, R. reniformis and T. semipenetrans on Thompson seedlessgrape cultivar. He noticed wide fluctuations during the growing season. The population was highly increased during April, August and October. However, Ismail (1992) showed that M. incognita infecting Thompson seedless grape cultivar has two periods of increase, the first

was during end of summer and early autumn, while the second was in late winter. Generally, the remarkable increase in soil population of the major genera of phytonematodes during spring is concomitant with the growing season of grapes and flush of new feeder roots. At that time of the year occur the abundance of succulent roots which are preferable for nematode infection. McKenry (1984)

Numbers in 250 g

soil

Numbers in 250 g

soil

Numbers in 250 g

soil

Numbers in 250 g

soil

Temper

ature °C

El-Marzoky, et al.

Fig. 2. Seasonal fluctuations behavior of four phytonematodes infecting King Ruby grapevine cultivar grown in sandy loam soilin relation to temperature at Sharkia Governorate

Dec.

2011

Jan.

2012

Feb.

2012

Mar.

2012

Apri

l 2012

May 20

12

June

2012

July

2012

Aug.

2012

Sept

. 2012

Oct.

2012

Nov.

2012


showed that in the spring root flush provided approximately 2-7root initials to each kg of moist soil or 30000 new roots per vine. On the other hand, thenematodes develop and reproduce faster at that time of the year whereas temperature begin to increase and encourage build up of soil population of phytonematodes.Spatial Distribution of CommonPhytonematodes Infecting FlameGrapevine Cultivar

The spatial pattern of plant-parasitic nematodes in an agricultural or natural ecosystem has two major components. The first one is thehorizontal distribution while the second is the vertical distribution throughout the soilor tillage (Been and Schomaker, 2006).

Regarding horizontal distribution, data in Table 1 and Fig. 3 indicated that population density of the root-knot nematode, M. incognita and thereniform nematode, R. reniformis were relatively higher as compared to lower values for T. semipenetrans, Tylenchorhynchus spp. and Helicotylenchus spp. General means of population density for M. incognita, R. reniformis, T. semipenetrans, Tylenchorhynchus spp. and Helicotylenchus spp. were 82.75,103.70, 22.30, 30.75 and 25.75 individuals per 250 g soil, respectively. Relative population density for these

nematodes averaged 31.20, 39.09,8.41, 11.59 and 9.71%, respectively. On the other hand,it was clear that total numbers of the detected nematodes were significantly differed (P≤ 0.05)from distance to another. For instances, within row total numbers of nematode species per 250 g soil at distances of 0-50 and 51-100 cm far from tree trunk were 352.5 and 149 individuals respectively, with percent distribution of 26.58 and 11.23%, respectively when compared with total numbers of nematodes counted in other investigated distances. The parallel values for M. incognita and R. reniformis were 141.75 (34.26%) and 35.00 (8.46%) in distance 0-50 cm as well as 147.50 (28.45%) and 76.75 (14.80%) in distance 51-100 cm. Regarding, total population density of all nematodes at distances of 0-50 , 51-100 and 101-150 cm from tree trunk between rows, it was found that,the highest value (491.5) was detected at the distance of 51-100 cm compared to moderate (219.00) or lower (114.25) values at distances of 0-50 and 101-150 cm, respectively. Percent distribution of total nematodes at distances of 0-50, 51-100 and 101-150 cm were 16.51,37.06 and 8.62%, respectively. The parallel values for M. incognita and R. reniformis were 9.00 (15.72%), 41.75 (33.03%) and 6.53 (8.00%), respectively.

El-Marzoky, et al.

Generally it could be concluded that in row between vines the nearest distance 0-50 cm contained the highest nematode numbers, whereas the distance of51-100 cm from tree trunk between rows showed the highest values of nematode numbers.

Concerning vertical distribution, data in Table 2 showed that general means of population density for M. incognitaand R. reniformis were higher as compared to other nematodes, since values of general means for M. incognita, R. reniformis, T. semipenetrans, Helicotylenchus spp. andXiphinema spp. were 125.00, 153.75, 9.00, 13.63 and 2.50 individuals per 250 g soil with relative population density of 41.14, 50.60, 2.96, 4.48 and 0.82%, respectively. On the other hand, total numbers of thecounted nematodes at 0-20 and 21-40 cm depths were 377.0 and 230.75 individuals per 250 g soil, with percent distribution of 62.03 and 37.97%, consecutively. This conclusion was true for all investigated nematodes except Xiphinema spp.

Population density of M. incognita,R. reniformis, T. semipenetrans and Helicotylenchus spp. were 157.25 (92.75), 184.25(123.25) , 14.25 (3.75) and 21.00 (6.25) nematodes at 0-20 and 21-40 cm , while the parallel values for Xiphinema spp.were 0.25 and 4.75 nematodes per250 g soil, respectively. Percent distribution for M. incognita, R. reniformis, T. semipenetrans, Helicotylenchus spp. and Xiphinema spp. at the two depths were 62.90 (37.10), 59.92(40.08), 79.17 (20.83), 77.06 (22.94) and 5 (95)%, respectively.

Many workers reported that spatial distribution of plant-parasitic nematodes is largely dependant on root distribution. Moreover, most reports on sampling and population estimation showed that the highest nematode densities occurred in the upper 15-20 cm of soil (Norton, 1978; Bird and Ramsdell, 1985; McSorley, 1987; Been and Schomaker, 2006; Padasaini et al., 2006). On the other hand, there were many exceptions, for example, Quader et al. (2003) in a South Australian vineyard revealed that Xiphinema spp.

Table 1. Horizontal distribution of common phytonematodesinfecting Flame grapevine cultivar at Sharkia Governorate

Distance from tree trunk (cm)

M. i

ncog

nita

R. re

nifo

rmis

T. se

mip

enet

rans

Tyle

ncho

rhyn

chus

spp.

Helic

otyl

ench

usspp.

Total No. of

nematodes

and

percent

distribution


Within rows 0-50 141.75

b147.50

ab22.25 b26.75 c 14.25

cd352.5 b(26.58%)

51-100 35.00 c 76.75 c 12.00 c

15.75de 9.50 e 149 d

(11.23%)Between rows 0-50 37.25 c 81.50 c 31.75 a33.00 b 35.5 b 219 c

(16.51%) 51-100 172.75

a171.25

a 33.25 a60.75 a53.50 a 491.5 a(37.06%)

101-150 27.00 d 41.50 d 12.25 c17.50 d16.00 c 114.25 e(8.62%)

Total No. of nematodes 413.75 518.50 111.50 153.75 128.75 1326.25

Relative population density (%) 31.20% 39.09% 8.41% 11.59% 9.71%General mean

82.75 B103.70

A 22.30 D30.75 C25.75 D 265.25* Each value represents mean of 5 replicates for population density per 250

g soil.** Means followed by the same lowercase letter in columns or uppercase

letter in row are not significantly different at P≤0.05 according toDuncan's multiple range test.

Fig. 3. Percent distribution of the common phytonematodes infesting grapevine orchard at Sharkia Governorate at five horizontal distances situated in rows (0-50 and 51-100 cm) and inter-rows (0-50, 51-100 and 101-150 cm) far from vine trunk

Percent of nematode

genera in each level

spp.

spp.

0-50 cm within row

0

5

10

15

20

25

30

35

40

45

50

M. incognita R. reniformis T. semipenetrans Tylenchorhynchus spp. Helicotylenchus spp.

Nem atode genera

Percent o

f nem

atod

e genera

in ea

ch leavel

51-100 cm within row

0-50 cm between rows 51-100 cm betweenrows

101-150 cm betweenrows

El-Marzoky, et al.

Table 2. Vertical distribution of common phytonematodes infectingFlame grapevine cultivar at Sharkia Governorate

Depth (cm)

M. i

ncog

nita

R. re

nifo

rmis

T. s

emip

enet

rans

Helic

otyl

ench

ussp

p.

Xiph

inem

a sp

p.

Tota

l No

. of

nema

tode

and

perc

ent

dist

ribu

tion

0-20 157.25a

184.25a

14.25 a21.00 a 0.25 b 377 a(62.03%

) 21-40 92.75

b123.25ab

3.75 b 6.25 ab 4.75 a 230.75b

(37.97%)

Total No. of nematodes 250.00 307.5 18.00 27.25 5.00 607.75Relative population density (%)

41.14% 50.60% 2.96% 4.48% 0.82%

General mean 125 B 153.75A

9.00 D 13.63 C 2.50 E 303.88


* Each value represents mean of 5 replicates for population density per 250g soil.

** Means followed by the same lowercase letter in columns or uppercaseletter in row are not significantly different at P≤0.05 according toDuncan's multiple range test.

occurred mainly along the rowand inter-rows with higherdensity at 30-60 cm depth.Moreover, Villate et al. (2008)showed that the highest numbersof X. index occurred at 40-110 cmdepth in French vineyard.

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