6 Infection of plant-parasitic nematodes by Paecilomyces.pdf

Infection of plant-parasitic nematodes by Paecilomyces

lilacinus and Monacrosporium lysipagum

Alamgir KHAN1,4,*, Keith L. WILLIAMS1 andHelena K.M. NEVALAINEN2,3

1Proteome Systems Ltd., 1/35-41 Waterloo Road, North Ryde, NSW, 2113, Australia;2Department of Chemistry and Biomolecular Sciences, Macquarie University,

Sydney, NSW, 2109, Australia; 3Macquarie University Biotechnology Institute,Macquarie University, Sydney, NSW, 2109, Australia; 4Australian ProteomeAnalysis Facility, Macquarie University, Level 4, Building F7B, Research

Park Drive, Sydney, NSW, 2109, Australia*Author for correspondence; e-mails: [email protected];[email protected]

Received 7 April 2005; accepted in revised form 19 October 2005

Abstract. Studying the mode of infection of a biocontrol agent is important in order toassess its efficiency. The mode and severity of infection of nematodes by a soil sapro-

phyte Paecilomyces lilacinus (Thom) Samson and a knob-producing nematode trappingfungus Monacrosporium lysipagum (Drechsler) Subram were studied under laboratoryconditions using microscopy. Infection of stationary stages of nematodes by P. lilacinus

was studied with three plant-parasitic nematodes Meloidogyne javanica (Treub) Chit-wood, Heterodera avenae Wollenweber and Radopholus similis (Cobb) Thorne. Paecil-omyces lilacinus infected eggs, juveniles and females of M. javanica by direct hyphalpenetration. The early developed eggs were more susceptible than the eggs containing

fully developed juveniles. As observed by transmission electron microscopy, fungalhypha penetrated the M. javanica female cuticle directly. Paecilomyces lilacinus alsoinfected immature cysts of H. avenae including eggs in the cysts and the eggs of R.

similis. Trapping and subsequent killing of mobile stages of nematodes by M. lysipagumwere studied with the above three nematodes. In addition, plant-parasitic nematodesPratylenchus neglectus (Rensch) Chitwood and Oteifa and Ditylenchus dipsaci (Kuhn)

Filipjev were tested withM. lysipagum. This fungus was shown to infect mobile stages ofall the plant-parasitic nematodes. In general, juveniles except those of P. neglectus, weremore susceptible to the attack than adults.

Key words: Ditylenchus, fungal infection, Heterodera, Meloidogyne, Monacrosporium,Paecilomyces, Pratylenchus, Radopholus, SEM, TEM

Abbreviations: DIC – differential interference contrast; LM – light microscope; PCA –potato carrot agar; PDA – potato dextrose agar; SC – surface coat; SEM – scanning

electron microscope; TEM – transmission electron microscope

BioControl (2006) 51:659–678 � IOBC 2006DOI 10.1007/s10526-005-4242-x

Introduction

The common soil-inhabiting fungus Paecilomyces lilacinus (Thom)Samson is an effective opportunistic parasite of nematode eggs (Jatalaet al., 1980; Villanueva and Davide, 1984; Davide and Zorilla, 1985).Paecilomyces lilacinus, particularly the strain 251, is currently used asa biological control agent against various plant-parasitic nematodes(Kiewnick et al., 2002; Brand et al., 2004; http://www.prophyta.com).The mode and severity of infection of different developmental stagesof nematodes vary according to specific fungal isolates and nematodespecies. Studies with the Peruvian isolate of P. lilacinus showed thatMeloidogyne incognita (Kofoid and White) Chitwood eggs of earlyembryonic developmental stages (prior to gastrulation) were moresusceptible to infection than the eggs containing fully developed juve-niles (Jatala et al., 1979; Dunn et al., 1982; Jatala, 1986). On theother hand, the Alabama isolate of P. lilacinus was capable of infect-ing M. arenaria (Neal) Chitwood eggs ranging from the 2–8-cellembryonic stages to fully developed juveniles (Morgan-Jones et al.,1984). Additionally, substantial variability in virulence among variousisolates of P. lilacinus was observed in an in vitro test using eggs andegg masses of M. incognita race 3 (Santos et al., 1992).

Paecilomyces lilacinus has also been reported to infect female nema-todes of Meloidogyne spp. and cysts of Heterodera spp. and Globoderaspp. (Jatala et al., 1979). In these cases, the fungal hyphae penetratednematodes mainly through body openings (Jatala, 1986); no informa-tion is available on the potential direct penetration of nematode cuti-cle. Holland et al. (1999) showed that the P. lilacinus strain 251produced infection pegs prior to penetration of M. javanica eggs; how-ever, the study was not extended to any other nematode species. Otherthan this, there is no additional information available on the detailedmechanism of infection of P. lilacinus strain 251 which is somewhatunexpected considering its importance as a biological control agent.

In contrast to P. lilacinus, which infects stationary stages of nema-todes by producing an infection peg, the nematode-trapping fungusMonacrosporium lysipagum (Drechsler) Subram captures mobile stagesof nematodes using adhesive knobs (Wimble and Young, 1983; Rubner,1994, 1996; Saikawa and Kaneko, 1994; Saxena and Mittal, 1995).Adhesive knobs attach to nematodes irreversibly and nematodes are kil-led shortly after being trapped. It has been suggested that the trappingstructures of predacious fungi may also produce toxin(s) that immobi-lize nematodes before fungal hyphae penetrate the cuticle (Olthof and

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Estey, 1963). Modes for capture and infection of plant parasitic nema-todes by nematode trappers, in particular Arthrobotrys spp. (Tunlidet al., 1992a) and Dactylella spp. (Wimble and Young, 1983, 1984) havebeen described. However, there is only limited information available onthe mechanisms of nematode capture used by Monacrosporium species.Published examples include studies into capturing of a free-living nema-tode Panagrellus redivivus (Linne) Goodey by Monacrosporium spp.(Saxena and Mittal, 1995). Also, variability in the virulence among iso-lates of M. ellipsosporum (Grove) Cooke & Dickinson was observedusing the M. incognita race 3 (Santos et al., 1992).

The work presented in this manuscript is a part of a larger projectfocused on studies into the infection processes of P. lilacinus and M.lysipagum (Khan et al., 2003a, b, 2004). Here we first describe themode of infection of the stationary stages of M. javanica, Heteroderaavenae Wollenweber and Radopholus similis (Cobb) Thorne in labora-tory conditions by P. lilacinus 251 using microscopy. The effect of M.lysipagum knob attachment was studied microscopically and in vitro.Our special interest was to explore modes of capture and effectivenessof capturing and killing of various plant-parasitic nematodes by set-ting up a screening process with the strain M. lysipagum IMI 375301,isolated from an egg mass of M. javanica on tomato in a glasshouseof the Macquarie University, Sydney. Following knob attachment,subsequent killing of two additional plant-parasitic mobile nematodesPratylenchus neglectus (Rensch) Chitwood and Oteifa, and Ditylen-chus dipsaci (Kuhn) Filipjev was assessed to quantify the infection.

Materials and methods

Cultures of fungi

Paecilomyces lilacinus strain 251 (deposited at National MeasurementInstitute (NMI) formerly known as Australian Government Analyti-cal Laboratory (AGAL), accession number 89/030550) culture wasmaintained on potato dextrose agar (PDA, Difco, MI, USA) andMonacrosporium lysipagum (IMI 375301) culture was maintained onpotato carrot agar (PCA) plates (Khan et al., 2006).

Cultures of nematodes

Cultures of M. javanica, H. avenae and R. similis have been describedby Khan et al., (2006). Pure cultures of P. neglectus and D. dipsaci

INFECTION OF PLANT-PARASITIC NEMATODES 661

(carrot culture) were kindly supplied by Dr. Maria Scurrah (DARDI,Plant Pathology Center, Plant Pathology Unit, Adelaide, SouthAustralia) and used from the supplied cultures.

Infection of second, third and fourth stage juveniles, females and eggmasses of M. javanica, and immature cysts of H. avenae by P. lilacinus

Swollen second stage (J2), third stage (J3) and fourth stage (J4) juve-niles (Eisenback and Triantaphyllou, 1991) and immature femaleswere extracted from galls (splitting the galls), and egg masses werepicked with fine forceps from a routine culture of M. javanica on to-mato. Immature cysts (white) were collected from a routine culture ofH. avenae on barley. Different developmental stages of juveniles (J2,J3 and J4), females and egg masses of M. javanica, and white cysts ofH. avenae were placed at the edge of 5-d-old colonies of P. lilacinusgrown on water agar plates (15 g/l). The plates were incubated at26±1 �C for various times for M. javanica (see Figure legends) and 10 dfor H. avenae. After incubation, egg masses and cysts were collectedfrom the plates and placed on a slide using a drop of lactophenol cottonblue (Daykin and Hussey, 1985) for microscopic examination. The in-fected developing juveniles of M. javanica were observed using lightmicroscope (LM) and infected females were observed using LM, scan-ning electron microscope (SEM) and transmission electron microscope(TEM). Egg masses were squashed before placing on a slide. InfectedH. avenae cysts were observed using LM.

Infection of R. similis by P. lilacinus

About 20–30 R. similis nematodes from an axenic culture were usedto make up a fresh culture on sterilized carrot pieces (approximately1 cm�1.5 cm wide and 1.5 mm thick) and incubated at 26±1 �C for40 d before transferring the carrot pieces onto 5-d-old colonies of P.lilacinus grown on water agar at 26±1 �C. Incubation was continuedfor 3 weeks at 26±1 �C to observe the infection of R. similis eggs byP. lilacinus. When the carrot pieces were covered with P. lilacinus hy-phae, the nematode eggs were extracted by splitting the carrot pieceson a glass slide with forceps. A drop of lactophenol cotton blue wasplaced on the carrot pieces and covered with another glass slide fol-lowed by examination using LM. To observe the infection of variouslife stages of R. similis, the nematode suspension (eggs, juveniles andadults) was inoculated onto 7-d-old colonies of P. lilacinus growing


on sterile dialysis membranes (Holland and Williams, 1996). Theplates were incubated at 26±1 �C for up to 3 weeks and infected eggsand nematodes were examined using LM.

Killing of mobile nematodes by M. lysipagum

The following experiments were carried out as part of a screeningprocess to explore the efficiency of M. lysipagum in the killing of mo-bile stages of various plant parasitic nematodes in vitro. To observethe killing of M. javanica juveniles by M. lysipagum, either 100 ll ofjuvenile suspension or an egg mass containing various developmentalstages of eggs was placed on 15-d-old colonies of the fungus grownon PCA plates at 21±1 �C. In order to produce H. avenae juveniles,approximately 25 cysts were incubated in 2 ml of water for 2 weeks atroom temperature (21±1 �C) to hatch out juveniles. A small amount(100 ll) of each H. avenae juveniles and mixed populations (juvenilesand adults) of mobile plant-parasitic nematodes R. similis, P. neglec-tus and D. dipsaci were inoculated on 15-d-old colonies of M. lysipa-gum grown on PCA plates. The plates were incubated at 21±1 �C inthe dark at various times for different nematodes (see Figure legends).Trapped and dead nematodes were counted and photographed underLM.

SEM and TEM observations of infected nematodes

After 4 d incubation with P. lilacinus, M. javanica female specimenswere fixed and examined using SEM as described by Holland et al.(1999). For TEM, the specimens were taken after 6 d incubation withP. lilacinus. The samples were fixed, post-fixed, dehydrated and ultrasections cut, stained and examined as described by Holland et al.(1999).

Results

Infection of second, third and fourth stage juveniles, females and eggmasses of M. javanica, by P. lilacinus

Paecilomyces lilacinus infected all life stages of M. javanica includingdeveloping eggs, juvenile containing eggs, juveniles and females underlaboratory conditions. Different developmental stages of juveniles (fromswollen J2 to J4) and immature females were infected by P. lilacinus


within 4 d exposure to the fungus on a water agar plate (Figure 1a).Within the 4 d exposure, mycelia from the infected juveniles producedconidiophores, which suggest that infection had occurred much earlier.Previously, we have seen that M. javanica eggs were infected by P. lilaci-nus after 1 d exposure to the fungus (Holland et al., 1999). As soon asthe fungus had proliferated in the developing juveniles, the juveniles be-came hyaline. Mature female nematodes were also highly susceptible byP. lilacinus and bore large amounts of conidiophores around the body(Figure 1b). All 20 females observed were infected. It appeared that thefungal hyphae had penetrated the female cuticle regardless of availabilityof any body openings. Scanning electron microscopic examination con-firmed that the fungus had proliferated throughout the whole body of afemale and not through the anus or vulva (A and V arrows in Fig-ure 2a). Transmission electron microscopic examination further con-firmed that P. lilacinus had directly penetrated through the cuticle of aM. javanica female (Figure 2b).

Ten infected mature females of M. javanica were squashed onslides. We found that P. lilacinus proliferating inside the female hadinfected most of the unlaid eggs and consumed the egg contents in allcases. An example is given in Figure 3a. Several infected eggs andjuveniles from an egg mass displayed disrupted structural elements

Figure 1. Infection of various developmental stages of juveniles including swollen sec-ond stage (J2), third stage (J3) and fourth stage (J4) juveniles, immature female andfemale (a) and a female (b) of Meloidogyne javanica by Paecilomyces lilacinus. (a) The

developing second stage juveniles and an immature female were highly infected after4 d incubation at 26±1 �C. Paecilomyces lilacinus proliferated in the juveniles and re-grew from the body to the outside and produced conidiophores. All the juveniles ex-

cept females became hyaline. F=female, C=conidiophore, IF=immature female. (b)Female was highly infected with P. lilacinus with a large number of condiophoresradiating from the female body. The photographs were taken using a phase contrast

microscope. The bar represents 200 lm.


(Figure 3b). Healthy eggs in an egg mass not exposed to fungus (con-trol) are shown in Figure 3d; well developed juveniles are clearly visi-ble in the eggs. When the early developmental eggs of M. javanicawere infected, the fungus consumed the egg contents and the hyphaegrew out from the egg (Figure 3c). These hyphae then infected othereggs in the vicinity or produced conidiophores. Some eggs were founddevoid of juveniles but hyphae were present in the eggs (Figure 3b).

Although eggs in the early embryonic developmental stages in anegg mass were infected by P. lilacinus more often than fully developedjuveniles in eggs (data not shown), eggs that contained juveniles werealso efficiently infected by the fungus (Figure 4). These juvenilesshowed various degrees of deformity and abnormal development asP. lilacinus utilized them as a substrate, and were eventually killed byP. lilacinus (Figure 4a, c). The juveniles in the eggs were motionlessand therefore considered dead. In the case of severe infection, theexternal cortical layer of the unhatched juveniles was split (Figure 4c).Juveniles in eggs that were not exposed to the fungus (control) devel-oped and hatched normally (Figure 4d). Juveniles inside the eggs werealso found covered with mycelia but it was not confirmed from a lightmicroscopic observation whether the juvenile was actually infected byhyphal penetration (Figure 4b).

Figure 2. Scanning (a) and transmission (b) electron micrographs show females ofMeloidogyne javanica infected by Paecilomyces lilacinus. The SEM image shows thatP. lilacinus infected the M. javanica female without penetrating through the anus orvulva (arrows A and V). The box (C) shows where the fungus produced conidio-

phores. The TEM image shows the direct penetration of P. lilacinus hypha (arrow H)through the M. javanica female cuticle (arrow C). The fungus and the nematode wereincubated together for 4 d for SEM and 6 d for TEM at 26±1 �C. The TEM photo-

graph was taken at 890,000� magnification.


Infection of H. avenae by P. lilacinus

Immature white cysts of H. avenae were highly infected by P. lilacinus(Figure 5a). Conidiophores and hyphae were found all around thecysts and P. lilacinus appeared to have penetrated the cyst surface ina random fashion also seen with M. javanica females (Figure 1b). Theeggs in the infected cysts were also severely infected by P. lilacinus

Figure 3. Infection of various developmental stages of Meloidogyne javanica eggs byPaecilomyces lilacinus. (a) shows a squash of an infected egg from an infected female.

Paecilomyces lilacinus consumed the egg contents. The female body is full of fungalhyphae (arrow H) and the fungus has consumed all the body contents (circled). (b)shows various developmental stages of eggs from an egg mass of M. javanica infected

by P. lilacinus. Infected eggs were full of fungal hyphae (arrow H), which consumedthe egg contents within 10 d of incubation at 26±1 �C. Juveniles appeared sick andtheir body was vacuolated (arrow J2). Healthy juveniles were also present (arrow HJ)

in the same infected egg mass. (c) shows an early embryonic stage egg of M. javanicainfected by P. lilacinus. There was no sign of a developing juvenile in the egg there-fore it is assumed that the egg was in an early developmental stage. This image sug-

gests that P. lilacinus does not have any preferences as to the place for thepenetration or growing out from the egg of M. javanica. The image shows P. lilacinushyphae growing out from the egg (arrow H). ES=eggshell. (d) shows control eggsfrom an egg mass not exposed to the fungus. Healthy and well developed juveniles

are present in the eggs. The photographs (a), (c) and (d) were taken using differentialinterference contrast (DIC) microscope and (b) using a phase contrast microscope.The bar represents 30lm (a) and (c) and 100lm (b) and (d).


(Figure 5b). Mature tanned cysts of H. avenae were not infected byP. lilacinus.

Infection of R. similis by P. lilacinus

More than 70% of the 380 R. similis eggs studied did not developfurther after being in contact with P. lilacinus hyphae (data notshown). Rather than exhibiting signs for direct penetration of the fungalhyphae, the eggs or other life stages of R. similis displayed abnormalgrowth when exposed to P. lilacinus. In some cases, R. similis eggs weredirectly infected by penetration of P. lilacinus hyphae after a prolongedexposure to the fungus (2–3-weeks). Fungal hyphae consumed the egg

Figure 4. Infection of Meloidogyne javanica juveniles in eggs by Paecilomyces lilacinus.The eggs were exposed to P. lilacinus for 6 d at 26±1 �C. (a) shows the disrupted body

of an encapsulated juvenile of which the cuticular layer was degraded. The fungal hy-phae covered the body and killed the juvenile. ES=eggshell, H=fungal hyphae,J2=juvenile. (b) shows a fully developed encapsulated juvenile of M. javanica covered

with P. lilacinus mycelia. ES=eggshell, H=fungal hyphae, J2=juvenile. (c) shows ajuvenile of M. javanica infected by P. lilacinus with structural elements disrupted. Thefungus disrupted the cuticular layer of the juvenile. J2=juvenile, H=fungal hyphae,

B=broken cuticle of juvenile. (d) shows a fully developed juvenile containing egg notexposed to the fungus (control). The photographs were taken using a DIC microscope.The bar represents 30lm (a), 50lm (b) and (c) and 20lm (d).


contents in the infected R. similis eggs (Figure 6a). The degree of hyphalproliferation in the infected eggs of R. similis (Figure 6a) was less thanin the infected eggs of M. javanica and H. avenae (Figures 3c and 5b,respectively). Occasionally, adult R. similis was also infected by P. lilaci-nus; however, it is not known whether the nematode was alive at thetime of infection (Figure 6b).

Figure 5. Infection of an immature white cyst of Heterodera avenae and an egg fromthe same infected cyst by Paecilomyces lilacinus. The cyst was exposed to P. lilacinusfor 10 d at 26±1 �C. In (a), the conidiophores were present around the cyst indicat-ing that P. lilacinus either penetrated into the cyst or grew out of the cyst at many

places. The head end (arrow h) of the cyst did not appear as an avenue for fungalinfection. In (b), an infected egg obtained from an infected cyst. Paecilomyces lilaci-nus proliferated within the egg and consumed the egg contents. The photographs

were taken using a dissecting (a) and DIC (b) microscopes. C=conidiophores ofP. lilacinus, ES=eggshell, and H=fungal hyphae. The bar represents 200lm (a) and30lm (b).


Killing of mobile nematodes by M. lysipagum

Just less than 100% (334 of the 336) hatched out M. javanica juve-niles studied were captured by the knobs of M. lysipagum and killedwithin 24 h of exposure to the fungus. The dead juveniles were foundaggregated on a PCA plate where a large number of knobs were pres-ent (Figure 7a). It appeared that the M. javanica juveniles were at-tracted to the M. lysipagum knobs. Freshly hatched juveniles of M.javanica were immobilized within 30 min after attachment of knobs tothe cuticle. Monacrosporium lysipagum hyphae grew all over the in-fected juveniles. The fungus consumed the body contents of infectedjuveniles within 7 d resulting in a transparent body (Figure 7b). Inmany cases, more than one knob was attached to a juvenile (Fig-ure 7c) causing rapid killing. During the struggling of trapped nema-todes, some knobs were detached from the fungal hyphae butremained attached to the nematodes. These knobs germinated as trophichyphae and penetrated the nematode body (Figure 7d).

In contrast to M. javanica juveniles which were attached to the M.lysipagum knobs within minutes, H. avenae juveniles were attachedafter a 2-d exposure to the fungus. Monacrosporium lysipagum hyphae

Figure 6. Infection of eggs and an adult Radopholus similis by Paecilomyces lilacinus.

The egg in (a), and eggs and adult in (b) were exposed to P. lilacinus for 14 d and21 d respectively, at 26±1 �C. In (a), fungal hyphae have grown into the egg (arrowH) and essentially consumed the egg contents. In (b), at the tail end (arrow T) of the

nematode, a bunch of fungal hyphae are either penetrating in or growing out fromthe nematode indicating that P. lilacinus used the natural openings of R. similis forpenetration. Hyphal penetration in R. similis might have occurred after death of thenematode. ES=eggshell, H=hyphae, and E=fungal infected (by hyphal penetration)

eggs. The photographs were taken using a polarizing microscope. The bar represents50lm (a) and 100lm (b).


penetrated the H. avenae juveniles and consumed the body contentscausing the body to become hyaline (Figure 8) similarly to that seenfor M. javanica juveniles. However, just less than 50% of the 150 H.avenae juveniles inoculated on a M. lysipagum plate were infected andkilled 7 d after inoculation (data not shown).

Monacrosporium lysipagum was capable of infecting R. similis juve-niles and adults but eggs were not infected (Figure 9). From theobservation of 180 R. similis nematodes (a mixture of juveniles and

Figure 7. Trapping and killing of Meloidogyne javanica juveniles by Monacrosporiumlysipagum. The M. javanica juveniles were inoculated on a M. lysipagum growth plateand incubated for 24 h for (a) and (c) and 7 d for (b) and (d) at 21±1 �C. In (a), thefungus produced spores (circle SP) and adhesive knobs (circle K). J=dead juveniles,

H=hyphae. (b) shows mass killing of M. javanica juveniles by M. lysipagum on anagar plate. The fungus penetrated the juvenile and consumed the contents. In thehyaline juvenile, the fungal hyphae were clearly visible (arrow H). K=knobs of

M. lysipagum, J2=juveniles of M. javanica. (c) shows a juvenile of M. javanicacaught by two knobs of M. lysipagum. The free arrows indicate the point of theattachment of a knob on the cuticle. K=knob, H=fungal hypha, J2=juvenile of M.

javanica. (d) shows a free knob (arrow K) of M. lysipagum that germinated as a tro-phic hypha (arrow H) and penetrated the cuticle of a M. javanica juvenile. Occasion-ally, the nematode managed to free itself from the mycelium but the knob remainedattached to the cuticle. J2=juvenile of M. javanica. The photographs were taken

using dissecting (a), DIC (b), inverted phase contrast (c) and polarizing (d) micro-scopes. The bar represents 200 lm (a) and (c) and 100 lm (b) and (d).


adults), 90% of the total nematode population was killed by M. lysi-pagum after exposure of only 20 h to the fungus. Within this time,100 % juveniles were killed (Figure 10a). Females were more suscepti-ble to M. lysipagum than males.

The nematode P. neglectus was susceptible to M. lysipagum and81% of the 289 nematodes were infected and killed within 20 h ofexposure to the fungus. Both adult males and females of P. neglectuswere more susceptible to infection than juveniles (Figure 10b).

The nematode D. dipsaci was also susceptible to M. lysipagum ob-served from a population of 346 nematodes. Seventy-three percent ofthe nematodes were killed after 20 h and 90% after 44 h exposure tothe fungus (Figure 10c). At both exposure times, juveniles were moresusceptible to fungal infection than adults.

Discussion

We have shown that the P. lilacinus 251 strain is capable of infectingall stages of developing eggs of M. javanica including the eggs thatcontained well-developed juveniles (J1 and J2) and various stages ofjuveniles (swollen J2 to J4). This is in an agreement with the findingsof Morgan-Jones et al. (1984) who showed that all egg stages, ranging

Figure 8. Infection of a juvenile of Heterodera avenae by Monacrosporium lysipagum.

Two weeks after exposure at 21±1 �C, the fungus consumed the body contents, thejuvenile became hyaline and only fungal hyphae were visible (arrow H). h=head ofthe nematode, J2=juvenile of H. avenae. The photograph was taken using a polariz-ing microscope. The bar represents 100 lm.


from eggs in an embryonic stage to eggs containing fully developedjuveniles, were infected by an Alabama isolate of P. lilacinus.

Paecilomyces lilacinus strain 251 penetrated the M. javanica femalecuticle directly under laboratory conditions. This finding is in concertwith the observations of Morgan-Jones et al. (1984) with the Ala-bama isolate of P. lilacinus and Kim et al. (1992) who studied theArkansas fungus 18. However, these results are different to the find-ings by Jatala et al. (1979) and Jatala (1986) who reported that thePeruvian isolate of P. lilacinus infected M. javanica females throughnatural or broken nematode body openings only. We have shown ear-lier (Khan et al., 2003a, 2004) that a particular serine protease andchitinase enzymes purified from the P. lilacinus strain 251 degradedM. javanica eggshells by individual or combined treatment of the en-zymes. Therefore, one possible explanation for the differing observa-tions on female penetration between the Peruvian isolate and theother P. lilacinus strains studied and reported (Morgan-Jones et al.,1984) may lie in a different profile of enzymes facilitating direct pene-tration.

Figure 9. Infection of an adult Radopholus similis by Monacrosporium lysipagum. The

fungus proliferated in the nematode and consumed the body contents. The nematodeeventually was hyaline 3 weeks after exposure to the fungus at 21±1 �C and only thefungal hyphae were visible (arrow H). An egg (E) of R. similis next to the infected

adult was uninfected and the juvenile hatched normally, however, becoming eventu-ally trapped by the fungus. H=fungal hyphae in nematode, T=tail of the nematode.The photograph was taken using polarizing microscope. The bar represents 100 lm.


Meloidogyne javanica eggs of in the early developmental stageswere infected at a higher frequency than eggs containing juveniles. Ithas been suggested that juveniles in the eggs were not being infectedby P. lilacinus because of the lack of appropriate enzymes needed bythe fungus for the degradation of the nematode cuticle (Jatala et al.,1979; Stirling, 1991). Our investigation shows that P. lilacinus strain251 was able to infect different developmental stages of juveniles (Figure 1),encapsulated juveniles occasionally (Figure 4) and also penetrated thefemale cuticle directly (Figure 2). Therefore it is reasonable to assume that

Figure 10. Trapping and killing of different life stages of Radopholus similis, Pratylenchusneglectus and Ditylenchus dipsaci by Monacrosporium lysipagum. The numbers of nema-

todes tested were as follows: (a) 180 (R. similis), (b) 289 (P. neglectus) and (c) 346 (D. dips-aci). The nematodes were exposed to the fungus on a plate that was incubated at21±1 �C for 20 h in (a) and (b), and up to 44 h in (c).


the enzymes produced by P. lilacinus strain 251 (Khan et al., 2003a) are in-deed effective in hyphal penetration through the cuticle of juveniles and fe-males of M. javanica.

Infection of the cyst nematode H. avenae by P. lilacinus was inves-tigated at the light microscopic level only. It was found that P. lilaci-nus infected immature females and infection occurred all over the cyst(Figure 5). This appeared to be similar to the infection pattern seenfor M. javanica females (Figure 1; Holland et al., 1999). The numberof R. similis eggs directly infected by P. lilacinus appeared consider-ably lower when compared to the eggs of the other nematodes stud-ied. It was also noted that P. lilacinus did not proliferate in theinfected eggs of R. similis as it did in the eggs of M. javanica and H.avenae, even after a prolonged exposure. Differences in the eggshellcomposition (Bird and McClure, 1976; Wharton, 1980; Bird and Bird,1991) or egg content between different nematode species may contrib-ute to the efficiency and severity of infection. Experimental datadescribing the differences in total egg contents between nematode spe-cies is not currently available. Meloidogyne javanica lays eggs in agelatinous matrix, H. avenae eggs remain in the cysts and R. similislays eggs in the host roots that are not enclosed in a sac. Therefore,eggs from the different nematodes are exposed differently to the envi-ronment and may also differ in their content.

Occasionally, it was found that P. lilacinus penetrated the mobilestages (juveniles and adults) of R. similis. This may have involvedimmobilization of nematodes. In our earlier studies (unpublished), wefound that a supernatant of a P. lilacinus culture grown on a cerealsubstrate had a paralyzing effect on R. similis suggesting that the fun-gus may produce biologically active compound(s) capable of immobi-lizing this particular nematode, whereas M. javanica juveniles werenot affected.

Five plant-parasitic nematodes were tested on agar plates to studyattachment of M. lysipagum knobs to the different nematode speciesand to quantify infectivity. It was found that M. javanica, R. similis,P. neglectus and D. dipsaci were susceptible but the cereal cyst nema-tode H. avenae was less susceptible to M. lysipagum. Published litera-ture has shown that another cereal cyst nematode H. schachtiiSchmidt was also found to be less susceptible than M. javanica to twoknob-producing nematode trapping fungi Monacrosporium ellipsospo-rum (Grove) Subram and Monacrosporium cionopagum (Drechsler)Subram in soil condition (Jaffee and Muldoon, 1995). Studies withArthrobotrys spp. which traps nematodes using adhesive structures,


showed that H. schachtii was also less susceptible than M. javanica toA. haptotyla Drechsler and A. thaumasia (Drechsler) Schenck et al. insoil as well as on agar plates (Jaffee, 1998). This may at least partlybe explained by differences in the cuticular surface coat (SC) of nema-todes (Spiegel and McClure, 1995). Jaffee and Muldoon (1995) haveshown that differences in the cuticular chemistry cause differences inthe attachment of adhesive structures to different nematode species.SC contains carbohydrates and glycoproteins (Reddigari et al., 1986;Spiegel and McClure, 1995) that provide targets for the attachment offungal lectins (Tunlid et al., 1992b). The SC composition varies be-tween nematode species and between nematode developmental stages(Spiegel and McClure, 1995). Additionally, the striation on the cuticleof H. avenae juveniles is stronger than in other nematode speciesstudied (Baldwin and Mundo-Ocampo, 1991). This stronger striationmay have resisted the attachment of knobs on the cuticle.

It was also observed that the level of knob attachment variedbetween juveniles, males and females in R. similis, P. neglectus andD. dipsaci. This may relate to the affinity of fungal lectins to the nem-atode SC, composition of which varies in different developmentalstages and nematode sexes. It has been reported that the juveniles ofa seed gall nematode Anguina tritici (Steinbuch) Filipjev exhibit moreaffinity for lectins than adults, whereas a greater diversity of lectinsbind to females of root-knot nematode M. javanica and M. incognita(Spiegel and McClure, 1995).

We noted that M. lysipagum knobs were attached more frequentlyat the head or tail end of the nematode (Figure 7) than other areas ofthe body. The surface carbohydrates are dispersed over the entirecuticle, however, they are specifically localized at the head or tail,thus providing a possible explanation for frequent attachment of fun-gal knobs to the cephalic region via lectins (Spiegel and McClure,1995).

It was also found in our studies that the free nematodes were at-tracted towards the nematodes caught by knobs on agar plates wherea huge number of fungal knobs were present. This suggested that achemical signal from the knob might be involved.

It can be concluded that P. lilacinus is able to infect all the lifestages of M. javanica and fungal hyphae penetrate the nematode cuti-cle directly. Paecilomyces lilacinus also infects eggs and immature cystsof H. avenae and eggs of R. similis. The knob-producing M. lysipagum ishighly capable of infecting mobile stages of plant-parasitic nematodes.


Acknowledgements

We thank Microscopy unit of the Department of Biological Sciences,Macquarie University, Sydney. Special thanks are due to Ron Oldfield,Debra Birch and Vienny Brown for skilful assistance in light and trans-mission electron microscopy and photo printing. Rita J. Holland isthanked for the continuous supply of P. lilacinus and M. javanica cul-tures and various discussions on experimental design and data collection.This work was funded by the Australian Post-graduate Award to AlamgirKhan.

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