Plant Disease / March 2001 277

Host-Parasite Relationships in Root-Knot Disease of White Mulberry

P. Castillo, Research Nematologist, Instituto de Agricultura Sostenible, Consejo Superior de Investigaciones Cien-tificas (IAS-CSIC), Apdo. 4084, 14080-Córdoba, Spain; M. Di Vito and N. Vovlas, Nematologist Research Lead-ers, Istituto di Nematologia Agraria, Consiglio Nazionale delle Ricerche, Via G. Amendola 165/a, 70126-Bari,Italy; and R. M. Jiménez-Díaz, Professor, IAS-CSIC and Escuela Técnica Superior de Ingenieros Agrónomos yMontes (ETSIAM), Universidad de Córdoba, Apdo. 3048, 14080 Córdoba

Severe feeder-root infections of gardengrown white mulberry trees and heavysoil infestations by Meloidogyne arenaria(Neal) Chitwood were found recently attwo localities in Córdoba and Sevillaprovinces in Andalucía, southern Spain.This is the first recorded infection ofwhite mulberry by this root-knot nema-tode in Europe. The highly devastatedroot system and the abundance of char-acteristic spherical galls suggested thatspecialized nematode-plant relationshipsmight occur.

White mulberry (Morus alba L.) is aperennial tree of the Moraceae family thatoriginates from the lower slopes of theHimalayas (17). This tree can grow underclimatic conditions ranging from temperateto tropical, and is economically importantin China, Egypt, and India, where the foli-age serves as food for the monophagoussilk worm (Bombix mori L.) (2,25,26). Inaddition, mulberry plantings play a role ina sustainable strategy for soil and waterconservation in north China (26).

Attacks by M. arenaria may be a con-straint for mulberry cultivation; however,little information exists on the host-parasite relationships between this root-knot nematode and white mulberry. M.arenaria is often found in greenhouses innorthern Europe, and is common in woodyfruit crops in European countries (12,14).The taxonomic identification of the M.arenaria population infecting these crops,including white mulberry, is of concernbecause this species is morphologicallyand geographically very close to M. his-panica Hirschmann. M. hispanica was firstdescribed parasitizing peach (Prunus per-sica L.) grown in Sevilla (8). In addition,populations of M. arenaria can be differ-entiated into two pathogenic races. Race 1reproduces on peanut (Arachis hypogaeaL.) and is distributed mainly in peanut-growing areas. Race 2 is widespread andcan reproduce on many hosts but not onpeanut and pepper (Capsicum annuum L.)(18). Thus, accurate identification of M.arenaria populations and their pathogenicrace characterization are needed for de-signing effective control measures in thecontext of sustainability and integrated pestmanagement. This is especially importantin root-knot nematodes, since host-plantresistance to reduce the initial nematodepopulation density is scarce among cropplants (18). Furthermore, the extent of cropgrowth impairment by the nematode is

influenced by nematode population density,with a minimum population density deter-mining the threshold for measurable yieldloss (the tolerance limit) (19). The objec-tives of this study were to determine: first,the taxonomic identity of the M. arenariapopulation infecting white mulberry; sec-ond, the histopathology in nematode-feeding sites on white mulberry roots; andthird, the relationship between initialpopulation density of the nematode andgrowth of white mulberry seedlings undergreenhouse conditions.

MATERIALS AND METHODSNematode diagnosis and pathogenic-

ity. The root-knot nematode infectingwhite mulberry was identified by means ofmicroscopic observations and isozymecharacterization of specimens (13). Sam-ples of white mulberry feeder roots to-gether with bulk soil were taken with ashovel from the upper 20 cm of soil fromeach of two home gardens at La Carlota(Córdoba province) and Utrera (Sevillaprovince) in southern Spain. Second-stagejuveniles and males extracted from rootsand soil (1), and females recovered frominfected root tissues, were mounted inglycerin. Nematode anatomy of glycerininfiltrated specimens were examined bylight microscopy. Single- or five-specimengroups of young egg-laying females werestudied by isozyme electrophoretic meth-ods (13).

For pathogenicity studies, the populationof root-knot nematodes collected frominfected white mulberry and a populationof M. hispanica infecting peach in Sevilla,Spain, were used for a comparative studyof M. arenaria-race differentials. Inoculumof M. arenaria consisted of eggs and sec-ond-stage juveniles collected with sodiumhypochlorite (9). Inoculum of M. hispanicawas first increased in tomato (Lycoper-sicum esculentum Mill. ‘Rutgers’) andextracted as that for M. arenaria. Twenty-day-old seedlings of cotton (Gossypiunhirsutum L. ‘Delta Pine’), peanut ‘Florun-ner’, pepper ‘Early California Wonder’,tobacco (Nicotiana tabacum L. ‘NC 95’),tomato ‘Rutgers’, and watermelon (Citrul-lus vulgaris Schad. ‘Charleston Grey’),were transplanted (one per pot) into 1-literclay pots filled with autoclaved field soil.Two days later individual seedlings wereinoculated by adding 10 ml of a suspension

ABSTRACTCastillo, P., Di Vito, M., Vovlas, N., and Jiménez-Díaz, R. M. 2001. Host-parasite relationshipsin root-knot disease of white mulberry. Plant Dis. 85:277-281.

Severe infections of white mulberry feeder roots and heavy soil infestations by Meloidogynearenaria race 2 were found in southern Spain. This is the first record of M. arenaria on whitemulberry in Europe. Morphometric observations, analysis of the esterase electrophoretic pat-tern, and artificial inoculations of race differentials were used to characterize nematodes.Nematode-induced mature galls were spherical and usually contained one or more females,males, and egg masses with eggs. Feeding sites were characterized by the development of giantcells that contained granular cytoplasm and many hypertrophied nuclei. Giant cell cytoplasmwas aggregated along a thickened cell wall. Vascular tissues within galls appeared disorganized.The relationship between the initial nematode population density (Pi) in a series from 0 to 1,024eggs and juveniles/cm3 soil and growth of white mulberry seedlings was tested in the green-house. A Seinhorst model was fitted to plant height and top fresh weight. Tolerance limits ofwhite mulberry to M. arenaria race 2 for plant height and top fresh weight were, respectively,1.1 and 1.38 eggs and juveniles/cm3 soil. The minimum relative values for plant height and topfresh weight were 0 at Pi > 64 and Pi > 128 eggs and juveniles/cm3 soil, respectively. Maximumnematode reproduction rate was 435-fold at the lowest Pi.

Additional keywords: histopathology, Morus alba, pathogenicity, threshold limit

Corresponding author: P. CastilloE-mail: ag1cascp@lucano.uco.es

Accepted for publication 12 November 2000.

Publication no. D-2001-0108-02R© 2001 The American Phytopathological Society

278 Plant Disease / Vol. 85 No. 3

containing 10,000 eggs and juveniles of theM. arenaria or M. hispanica populations.Plants that served as controls received thesame amount of water. Plants were incu-bated in a greenhouse adjusted to 26 ± 2ºC.There were four plants per nematodepopulation-race differential combination.Fifty days after inoculation plants wereuprooted and their roots gently washed,examined, and rated both for galls and eggmasses developed.

Histopathology. Galled roots from natu-rally M. arenaria-infected white mulberryplants sampled at La Carlota (Córdoba)and from plants artificially infected in theinoculum-density plant-growth experimentwere selected for histopathological studies.Roots were gently washed free of soil anddebris, and individual galls selected. Rootsegments of uninfected seedlings served ascontrol. Galled and healthy root tissueswere fixed in formaldehyde chromoaceticsolution for 48 h, dehydrated in a tertiarybutyl alcohol series (40-70-85-90-100%),and embedded in 58ºC-melting point paraf-

fin for histopathological observations.Embedded tissues were sectioned with arotary microtome. Sections 10 to 12 µmthick were mounted on glass slides, stainedwith safranin and fast-green, mountedpermanently in dammax xylene, examinedmicroscopically, and photographed (10).

Inoculum-density plant-growth im-pairment relationship. Inoculum of M.arenaria was extracted (9) from whitemulberry roots collected at La Carlota.This inoculum was increased in tomato‘Rutgers’ inoculated as before and incu-bated in a greenhouse adjusted to 26 ± 2ºC.Two months after inoculation, at the timethat egg masses were well formed in thetomato roots, the inoculated plant rootswere washed free of soil and finelychopped. To estimate the amount of eggsand juveniles formed in the chopped tissue,10 5-g aliquots of infected chopped rootswere suspended in 1% aqueous solution ofsodium hypochlorite in 100-ml jars for 4min (9). For inoculation, chopped infectedroots were thoroughly mixed with 3 kg of

steam-sterilized sandy soil and the mixturewas used as inoculum within a range ofinoculum densities. Appropriate amountsof this inoculum were mixed with a pottingmixture of 97.5% steam-sterilized sandysoil (sand 88%, silt 5%, clay 7%) and 2.5%organic matter, to reach a population

Fig. 1. Diagnostic features of Meloidogyne arenaria. A and B, Female anterior regions. C, D, and E, Female perineal patterns. F, G, and H, Second-stagejuvenile. I and J, Male anterior region. K, Lateral fields of male at mid-body. L, Male tail region; ep = excretory pore.

Fig. 2. Meloidogyne arenaria race 2. Esteraseelectrophoresis pattern of protein homogenatesfrom five (A3/a) and single young egg-layingfemales (A3/b). J3 = M. javanica (referencepopulation).

Plant Disease / March 2001 279

Fig. 3. Host-parasite relationships between Meloidogyne arenaria race 2 and white mulberry. A, and B, Galled roots from naturally infected plants, show-ing spherical galls. C, Longitudinal section of naturally infected roots showing giant cells and galling induced by M. arenaria. D, Display of plants artifi-cially inoculated with M. arenaria showing marked reduction in shoot growth. E, Root systems of plants artificially inoculated with 1,024 eggs + J2/cm3

of soil of M. arenaria (left) and uninoculated control (right), respectively. F, G, H, and I, Transverse sections of roots from plants artificially inoculatedwith M. arenaria. Abbreviations: gc = giant cell; h = hypertrophic nucleus; n = nematode; t = thickened cell wall; x = xylem.

280 Plant Disease / Vol. 85 No. 3

density of 0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16,32, 64, 128, 256, 512, and 1,024 eggs andjuveniles/cm3 soil, and 600-ml clay potswere filled with the infested soil mixture.A single, 2-month-old seedling of whitemulberry ‘Superior’ was transplanted intoeach pot. There were eight pots for eachinoculum density, arranged in a random-ized complete block design in a greenhouseat 26 ± 2ºC. Three months after trans-planting, top fresh weight and height ofplants were recorded. Plants were up-rooted, the roots washed free of adheringsoil and weighed, and eggs and juveniles inthe egg masses in roots were extracted bythe sodium hypochlorite method (9).Nematodes in soil were extracted by themodified Coolen’s method (1,4). The finalnematode population densities were calcu-lated as the total of that from roots andsoil. The relationship between plant growth(indicated by the top fresh weight andheight of plants) and the initial nematodepopulation density was determined byfitting the data to the Seinhorst model: y =m + (1 – m)zP–T when P > T, and y = 1when P < T (19,20). In this model, y =relative value of the plant growth parame-ter; m = minimum y value (y at a very largeinitial nematode population density); P =the initial nematode population density; T= a tolerance limit (initial population atwhich plant growth is not impaired) and zis a constant >1 reflecting nematode dam-age, with z–T = 1.05 (19). The Seinhorstequation was fitted using the SeinFit pro-gram (21). The coefficient of determina-tion (R2) and the residual sum of squareswere used to indicate goodness-of-fit ofdata to the model.

RESULTS AND DISCUSSIONNematode diagnosis and pathogenic-

ity. Detailed morphometric observationsbased on the sampled second-stage juve-niles, shape of male stylet knobs and fea-tures of the female perineal pattern (Fig. 1)agreed with those that characterize M.arenaria (15). The isozyme electrophoreticanalyses of single- and five-specimengroups of young egg-laying sampled fe-males (Fig. 2) revealed the esterase patternthat is characteristic of M. arenaria (6).Inoculations of the M. arenaria race differ-entials indicated that the population of theroot-knot nematode from white mulberryin southern Spain is not virulent to cotton,peanut, and pepper, but is virulent to to-bacco, tomato, and watermelon. Therefore,this population was identified as M. arena-ria race 2. The pattern of disease reactionsinduced by the population of M. hispanicain the differentials host was the same thanthat induced by M. arenaria. Therefore,these two species of root-knot nematodesare closely related both taxonomically andbiologically.

Histopathology. Galls occurred eithersingly or in clusters which encircled theentire root perimeter. In this latter case the

root diameter was 2 to 6 times larger thanthat of uninfected roots (Fig. 3). More than45% of individual galls selected at randomcontained an egg mass. Usually, galls con-tained more than one nematode female.Observations of stained root sections re-vealed both tissue hypertrophy and hyper-plasia, as well as disorganization anddisruption of xylem elements and primaryphloem cells. Nematode feeding sitescomprised 3 to 8 giant cells that sur-rounded the lip region of a single female.Undersized feeding cells were associatedwith pre-adult males. Active multinucle-ated giant cells contained granular cyto-plasm, thickened cell wall, and numeroushypertrophied nuclei and nucleoli. Densegiant cell cytoplasm lined deeply stainedthick walls. The histological and anatomi-cal changes induced by M. arenaria tomulberry roots were similar to those de-

scribed for other root-knot nematode spe-cies infecting fruit trees (11,14,22).

Inoculum-density plant-growth im-pairment relationship. The inoculumdensities of the nematode included in thestudy impaired growth of mulberry plants(Fig. 3). The relationship between the topfresh weight and height of plants and theinitial nematode population density wasappropriately described by the Seinhorstequation (Fig. 4). Symptoms of attack byM. arenaria race 2 and reduction of planttop growth were evident 10 days after in-oculation even with an initial populationdensity of Pi = 16 eggs and juveniles/cm3

soil. Reduction of plant top growth wasalso evident at lower initial populationdensities 20 days later. The white mulberrytolerance limits (T) to M. arenaria race 2were 1.1 and 1.38 eggs and juveniles/cm3

soil for height plant and top fresh weight,

Fig. 4. Relationship between initial population densities (Pi) of a population of Meloidogyne arena-ria race 2 from Spain and relative top fresh weight and height of white mulberry plants grown in potsat 26 ± 2°C in the greenhouse for 3 months. Actual data are presented for top weight (•) and plantheight ( ). Solid and dashed lines represent the predicted model. Statistics for fitted models of topand plant height were: R2 = 0.84, Sum of squares = 16.31; and R2 = 0.90, Sum of squares = 154.16,respectively.

Table 1. Relationship between initial population density of Meloidogyne arenaria race 2 (Pi, eggsand juveniles per cm3 soil) and final population density (Pf) and reproduction rate (Pf/Pi) in whitemulberry seedlingsa

Initial population density (Pi) Final population density (Pf) Reproduction rate (Pf/Pi)

0.125 20.6 164.80.25 108.7 434.80.5 135.2 270.41 99.2 99.22 91.8 45.94 68.4 17.18 86.7 10.8

16 42.2 2.632 20.8 0.664 15.1 0.2

128 50.6 0.4256 21.0 0.1512 26.9 0.1

1,024 23.1 0

a Two-month-old white mulberry seedlings were transplanted (one per pot) into a potting mixtureinfested with the appropriate Pi. Plants were grown in a greenhouse adjusted to 26 ± 2ºC for 3months.

Plant Disease / March 2001 281

respectively (Fig. 4). The minimum rela-tive value (m) for plant height and topfresh weight was 0 at Pi > 64 and Pi > 128eggs and juveniles/cm3 soil, respectively.The maximum nematode reproduction rate(Pf/Pi; Pi = initial population density, Pf= final popuation density) was 434.8 at Pi= 0.25 eggs and juveniles/cm3 of soil. Thisreproduction rate decreased as the initialnematode population increased (Table 1).Reduction of nematode reproduction ratewith increasing initial nematode inoculumdensity has been reported associated withinfections of several crops by M. incognita(3–5). Our findings could be a conse-quence of nematode competition for nutri-ents or root tissue availability (feedingsites), as a result of which a smaller pro-portion of the inoculum would developsuccesfully.

Results demonstrated that M. arenariarace 2 has the potential to severely impairgrowth of white mulberry. The tolerancelimit of this plant to the nematode is as lowas approximately 1 egg/cm3 soil. An initialpopulation density of this parasite exceed-ing 64 eggs/cm3 soil had a lethal result forwhite mulberry. Our results on patho-genicity of M. arenaria on white mulberryagreed with those of other researchers inChina (23,24) and India (7,16), who foundthat M. arenaria and M. incognita (Kofoidet White) Chitwood significantly reducedthe number of leaves and plant growth ofwhite mulberry trees. Therefore, controlmeasures need to be implemented in orderto guarantee production of nematode-freeplanting stocks and to avoid spread of thenematode to areas not infested.

ACKNOWLEDGMENTSWe thank G. Zaccheo and F. Catalano for their

technical assistance in conducting of the green-house experiments; D. Esmenjaud from INRA,Antibes (France) for kindly providing a samplepopulation of M. hispanica from roots of infectedtomato; I. de O. Abrantes and C. Santos, Departa-mento de Zoologia, Universidade de Coimbra,Portugal, for their help with the isozyme electro-

phoretic studies; and H. Rapoport from IAS-CSICfor her critical revision of the manuscript.

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