Isolation of nematophagous fungi from eggs and females of Meloidogyne spp. and evaluation of their biological control potential

Fungi were isolated from Meloidogyne spp. eggs and females on 102 field-collected root samples in China. Of the 235 fungi isolated (representing 18 genera and 26 species), the predominant fungi were Fusarium spp. (42.1% of the isolates collected), Fusarium oxysporum (13.2%), Paecilomyces lilacinus (12.8%), and Pochonia chlamydosporia (8.5%). The isolates were screened for their ability to parasitise Meloidogyne incognita eggs in 24-well tissue culture plates in two different tests. The percentage of eggs parasitised by the fungi, the numbers of unhatched eggs and alive and dead juveniles were counted at 4 and 7 days after inoculation. The most promising fungi included five Paecilomyces isolates, 10 Fusarium isolates, 10 Pochonia isolates and one Acremonium isolate in test 1 or test 2. Paecilomyces lilacinus YES-2 and P. chlamydosporia HDZ-9 selected from the in vitro tests were formulated in alginate pellets and evaluated for M. incognita control on tomato in a greenhouse by adding them into a soil with sand mixture at rates of 0.2, 0.4, 0.8 and 1.6% (w/w). P. lilacinus pellets at the highest rate (1.6%) reduced root galling by 66.7%. P. chlamydosporia pellets at the highest rate reduced the final nematode density by 90%. The results indicate that P. lilacinus and P. chlamydosporia as pellet formulation can effectively control root-knot nematodes.     

Association of Verticillium chlamydosporium and Paecilomyces lilacinus with Root-knot Nematode Infested Soil

Population densities of Meloidogyne incognita and the nematophagous fungi, Paecilomyces lilacinus and Verticillium chlamydosporium, were determined in 20 northern California tomato fields over two growing seasons. Paecilomyces lilacinus was isolated from three fields, V. chlamydosporium was isolated from one field, and both fungi were isolated from 12 fields. Verticillium chlamydosporium numbers were positively correlated with numbers of M. incognita and P. lilacinus. Paecilomyces lilacinus numbers were positively correlated with V. chlamydosporium numbers, but they did not correlate with M. incognita numbers. The correlation coefficients were low (R < 0.5) but significant (P < 0.05). All P. lilacinus and V. chlamydosporium field isolates parasitized M. incognita eggs in vitro. In a greenhouse study, numbers of V. chlamydosporium and P. lilacinus increased more in soils with M. incognita-infected tomato plants than in soil with uninfected tomato plants. After 10 weeks, the Pf/ Pi of second-stage juveniles in soils infested with P. lilacinus, V. chlamydosporium, and M. incognita was 47.1 to 295.6. The results suggest V. chlamydosporium and P. lilacinus are not effectively suppressing populations of M. incognita in California tomato fields.     

Biological control of root rot-root knot disease complex of tomato

Efficacy of Pseudomonas aeruginosa alone or in combination with Paecilomyces lilacinus was evaluated in the control of root-knot nematode and root-infecting fungi under laboratory and field conditions. Ethyl acetate extract (1 mg/ml) of P. lilacinus and P. aeruginosa,respectively, caused 100 and 64% mortality of Meloidogyne javanica larvae after 24 h. Ethyl acetate fractions of biocontrol agents were more effective than hexane extracts in the suppression of M. javanica larvae, indicating that active nematicidal compounds are intermediary in polarity. In field experiments, biocontrol fungus and bacterium significantly suppressed soilborne root-infecting fungi including Macrophomina phaseolina, Fusarium oxysporum, Fusarium solani, Rhizoctonia solani and Meloidogyne javanica, the root-knot nematode. P. lilacinus parasitized eggs and female of M. javanica and this parasitism was not significantly influenced in the presence of P. aeruginosa. P. aeruginosa was reisolated from the inner root tissues of tomato, whereas P. lilacinusdid not colonize tomato roots. This revised version was published online in June 2006 with corrections to the Cover Date.     

Histology of the Interactions of Paecilomyces lilacinus with Meloidogyne incognita on Tomato

Excised tomato roots were examined histologically for interactions of the fungus Paecilomyces lilacinus and Meloidogyne incognita race 1. Root galling and giant-cell formation were absent in tomato roots inoculated with nematode eggs infected with P. lilacinus. Few to no galls and no giant-cell formation were found in roots dipped in a spore suspension of P. lilacinus and inoculated with M. incognita. Numerous large galls and giant cells were present in roots inoculated only with M. incognita. P. lilacinus colonized the surface of epidermal cells as well as the internal cells of epidermis and cortex. The possibility of biological protection of plant surfaces with P. lilacinus against root-knot nematodes is discussed.     

Histology of the interactions of Paecilomyces lilacinus with Meloidogyne incognita on Eclipta alba (L.)

Abstract

Under greenhouse conditions, the experiment was conducted to test the efficacy of opportunistic and nematophagous hyphomycete, Paecilomyces lilacinus against root-knot nematode, Meloidogyne incognita on Eclipta alba. The treatment comprised inoculation of E. alba with M. incognita alone (N) and in combination with P. lilacinus (one week before (T₁), simultaneously (T₂), one week after (T₃), two weeks after (T₄) and three weeks after (T₅)). The results showed that the application of P. lilacinus before one week of nematode inoculation (T_l) was more effective than other treatments. A significant enhancement was noticed in the growth and yield of E. alba. Root-knot and egg mass indices were suppressed due to destruction of the mature females and the egg masses. Histopathological studies revealed the presence of P. lilacinus hyphae in and around the females and the eggs.     

Evaluation of fungal and bacterial bioagents against Rhizoctonia solani on Solanum melongena (L.)

Three fungal and one bacterial bioagents were tested for efficacy against Rhizoctonia solani under in vivo and in vitro conditions. Organisms including native isolates of Trichoderma virens 7109, Paecilomyces lilacinus 7115, Aspergillus niger and Pseudomonas strain were investigated in different combinations to get effective antagonists. T. virens 7109 and A. niger significantly reduced the growth of R. solani under laboratory and field conditions, respectively. Fungal combination had stimulatory effect on total fungal population and was recorded the highest (25.04 × 10⁴ cfu/g soil) in T. virens 7109 + P. lilacinus 7115 combination. Soil treatments with individual bioagents have shown uniformity in disease suppression when compared to combinations. Treatment with combination of bioagents yielded higher fruit harvest when compared to that with single bioagent.     

Viability of Heterodera glycines Exposed to Fungal Filtrates

Filtrates from nematode-parasitic fungi have been reported to be toxic to plant-parasitic nematodes. Our objective was to determine the effects of fungal filtrates on second-stage juveniles and eggs of Heterodera glycines. Eleven fungal species that were isolated from cysts extracted from a soybean field in Florida were tested on J2, and five species were tested on eggs in vitro. Each fungal species was grown in Czapek-Dox broth and malt extract broth. No toxic activity was observed for fungi grown in Czapek-Dox broth. Filtrates from Paecilomyces lilacinus, Stagonospora heteroderae, Neocosmospora vasinfecta, and Fusarium solani grown in malt extract broth were toxic to J2, whereas filtrates from Exophiala pisciphila, Fusarium oxysporum, Gliocladium catenulatum, Pyrenochaeta terrestris, Verticillium chlamydosporium, and sterile fungi 1 and 2 were not toxic to J2. Filtrates of P. lilacinus, S. heteroderae, and N. vasinfecta grown in malt extract broth reduced egg viability, whereas F. oxysporum and P. terrestris filtrates had no effect on egg viability.     

Infection of plant-parasitic nematodes by Paecilomyces lilacinus and Monacrosporium lysipagum

Studying the mode of infection of a biocontrol agent is important in order to assess its efficiency. The mode and severity of infection of nematodes by a soil saprophyte Paecilomyces lilacinus (Thom) Samson and a knob-producing nematode trapping fungus Monacrosporium lysipagum (Drechsler) Subram were studied under laboratory conditions using microscopy. Infection of stationary stages of nematodes by P. lilacinus was studied with three plant-parasitic nematodes Meloidogyne javanica (Treub) Chitwood, Heterodera avenae Wollenweber and Radopholus similis (Cobb) Thorne. Paecilomyces lilacinus infected eggs, juveniles and females of M. javanica by direct hyphal penetration. The early developed eggs were more susceptible than the eggs containing fully developed juveniles. As observed by transmission electron microscopy, fungal hypha penetrated the M. javanica female cuticle directly. Paecilomyces lilacinus also infected 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. lysipagum were studied with the above three nematodes. In addition, plant-parasitic nematodes Pratylenchus neglectus (Rensch) Chitwood and Oteifa and Ditylenchus dipsaci (Kuhn) Filipjev were tested with M. lysipagum. This fungus was shown to infect mobile stages of all the plant-parasitic nematodes. In general, juveniles except those of P. neglectus, were more susceptible to the attack than adults.     

Growth of Isolates of Paecilomyces lilacinus and Their Efficacy in Biocontrol of Meloidogyne incognita on Tomato

The potential of 13 Paecilomyces lilacinus isolates from various geographic regions as biocontrol agents against Meloidogyne incognita, the effects of temperature on their growth, and the characterization of the impact of soil temperature on their efficacy for controlling this nematode were investigated. Maximum fungal growth, as determined by dry weight of the mycelium, occurred from 24 to 30 C; least growth was at 12 and 36 C. The best control of M. incognita was provided by an isolate from Peru or a mixture of isolates of P. lilacinus. As soil temperatures increased from 16 to 28 C, both root-knot damage caused by M. incognita and percentage of egg masses infected by P. lilacinus increased. The greatest residual P. lilacinus activity on M. incognita was attained with a mixture of fungal isolates. These isolates effected lower root-galling and necrosis, egg development, and enhanced shoot growth compared with plants inoculated with M. incognita alone.     

In vitro studies of antagonistic fungi against the root-knot nematode,Meloidogyne incognita

The present study was carried out in vitro to determine the efficacy of indigenous fungi isolated from egg masses of root-knot nematode, Meloidogyne incognita on egg parasitism, egg hatching, mobility and mortality against root-knot nematode, M. incognita. The tested fungi were Acremonium strictum, Aspergillus terreus, A. nidulans, A. niger, Chetomium aubense, Chladosporium oxysporum, Fusarium chlamydosporium, F. dimarum, F. oxysporum, F. solani, Paecilomyces lilacinus, Pochonia chlamydosporia, Trichoderma viride and T. harzianum. All tested fungi showed varied effects against the nematodes. Culture filtrates of A. strictum was very effective against the nematode in regards to egg parasitism (53%), egg hatching inhibition (86%) and mortality (68%) compared to controls. A. strictum was found to have an advantage over P. lilacinus, P. chlamydosporia, T. viride and T. harzianum in that it caused greater mortality of the second stage juveniles (J₂). A. terreus did not show egg parasitism but was found to be highly toxic against second stage juveniles (J₂) causing high mortality (around 68%). Thus, A. strictum and A. terreus showed good biocontrol potential against root-knot nematode, M. incognita under in vitro conditions.     

Pathogenicity of Fungi to Eggs of Heterodera glycines

Twenty-one isolates of 18 fungal species were tested on water agar for their pathogenicity to eggs of Heterodera glycines. An egg-parasitic index (EPI) for each of these fungi was recorded on a scale from 0 to 10, and hatch of nematode eggs was determined after exposure to the fungi on water agar for 3 weeks at 24 C. The EPI for Verticillium chlamydosporium was 7.6, and the fungus reduced hatch 74%. Pyrenochaeta terrestris and two sterile fungi also showed a high EPI and reduced hatch 42-73%. Arthrobotrys dactyloides, Fusarium oxysporum, Paecilomyces lilacinus, Stagonospora heteroderae, Neocosmospora vasinfecta, Fusarium solani, and Exophiala pisciphila were moderately pathogenic to eggs (EPI was 2.0-4.5, and hatch was reduced 21-56%). Beauveria bassiana, Hirsutella rhossiliensis, Hirsutella thompsonii, Dictyochaeta heteroderae, Dictyochaeta coffeae, Gliocladium catenulatum, and Cladosporium sp. showed little parasitism of nematode eggs but reduced hatch. A negative correlation was observed between hatch and fungal parasitism of eggs. Fusarium oxysporum, H. rhossiliensis, P. lilacinus, S. heteroderae, V. chlamydosporium, and sterile fungus 1 also were tested in soil in a greenhouse test. After 3 months, the nematode densities were lower in soil treated with H. rhossiliensis and V. chlamydosporium than in untreated soil. The nematode population densities were correlated negatively with the EPI, but not with the percentage of cysts colonized by the fungi. Plant weights and heights generally increased in the soil treated with the fungi.     

Management of Root-Knot Nematode,Meloidogyne Incognita,by integration of Paecilomyces Lilacinus with organic materials in Chilli

Studies were made to determine the efficacy of Paecilomyces lilacinus in management of root-knot nematode (Meloidogyne incognita) in soil amended with various organic matters. The soil amendments with organic additives except gram and rice husks significantly reduced the multiplication of M. incognita and the root galling caused by root-knot nematode which consequently increased the plant growth. The greatest improvement in plant growth and reduced reproduction factor and root galling was recorded in soil amendment with leaves of Calotropis procera while the least was in kail saw dust. The best protection against M. incognita was observed on the integration of organic additives with P. lilacinus, which resulted increased plant growth and reduced population build-up of nematodes and root gallings. The leaves of C. procera with P. lilacinus were most effective than all other organic materials used among the different integrated approaches. The organic amendments also increased the parasitism of P. lilacinus on M. incognita.     

Effect of Population Dynamics of Pseudomonas cepacia and Paecilomyces lilacinus on Colonization of Polyfoam Rooting Cubes by Rhizoctonia solani

Suspensions of Pseudomonas cepacia (strain 5.5B) and Paecilomyces lilacinus (isolate 6.2F) were applied to polyfoam rooting cubes for control of stem rot of poinsettia caused by Rhizoctonia solani. The populations of antagonists and colonization of rooting cubes by R. solani were monitored during a 3-week period. Colonization of cubes by R. solani was reduced in cubes treated with P. cepacia, but the population of P. cepacia decreased by as much as 97% during the test period. Increased colonization by R. solani was correlated with a decline in population of P. cepacia. P. lilacinus was more persistent than P. cepacia in cubes, with only a 21% reduction observed during the 3-week period. Colonization of the P. lilacinus-treated cubes by R. solani was significantly less than colonization of infested controls. No correlation existed between population of P. lilacinus and colonization of cubes by R. solani.     

Studies on interaction of Meloidogyne incognita (Kofoid and White) Chitwood and Fusarium solani (Mart.) Sacc forming a disease complex in lentil (Lens culinaris Medik.)

Abstract

An experiment was conducted to study the effects of interaction between Meloidogyne incognita and Fusarium solani on plant length, fresh and dry weights, number of pods, chlorophyll, carotenoid, nitrogen and phosphorus contents and nitrate reductase activity in lentil plants. The results reveal a maximum damage occurring in all the plant growth, biochemical and nutrient parameters, in plants inoculated with M. incognita 10 days prior to F. solani (Mi?→?Fs). This was followed by simultaneous (Mi?+?Fs) inoculations, fungus inoculation 10 days prior to nematode (Fs?→?Mi), M. incognita alone and F. solani alone treatments. Nematode reproduction factor and root galling were highest in individual inoculation of M. incognita, while root rotting percentage was highest when nematode was inoculated 10 days prior to fungus followed by simultaneous inoculation with both nematode and fungus.     

Effects of crop plants on abundance of Pochonia chlamydosporia and other fungal parasites of root‐knot and potato cyst nematodes

The effects of a host plant on reproduction/abundance of fungal populations in relation to soil nutrients released by plants in the rhizosphere were studied. Abundance in the soil and potato rhizosphere of the fungi Paecilomyces lilacinus, Monographella cucumerina (CABI 380408) and Pochonia chlamydosporia var. chlamydosporia (Pc280, potato cyst nematode biotype) and P. chlamydosporia var. catenulata (Pc392, root‐knot nematode biotype) were assessed. The different ability of break crops (oilseed rape, sugarbeet and wheat) in the potato rotation to support Pa. lilacinus, Pochonia isolates Pc280 and Pc392 and abundance of the latter two isolates in soil and rhizosphere of potato plants infected with Meloidogyne incognita were also studied. Potato chits and crop seedlings were planted into boiling tubes containing 5000 chlamydospores or conidia g^?1 in acid washed sand (pH 6) and kept in a growth chamber at 20°C, and 16 h of light for up to 9 weeks. The abundance of the fungi in sand (fallow) differed significantly between fungal species, being in general less abundant in the absence than in the presence of the plant, although there was no interaction between plant species and fungal isolate. There was evidence of a different response to Me. incognita for Pc392 than for Pc280 but there was no significant effect of the presence of the nematode on the rate of increase of the fungus.     

Effects of Paecilomyces lilacinus protease and chitinase on the eggshell structures and hatching of Meloidogyne javanica juveniles

A serine protease and an enzyme preparation consisting of six chitinases, previously semi-purified from a liquid culture of Paecilomyces lilacinus strain 251, were applied to Meloidogyne javanica eggs to study the effect of the enzymes on eggshell structures. Transmission electron microscopic studies revealed that the protease and chitinases drastically altered the eggshell structures when applied individually or in combination. In the protease-treated eggs, the lipid layer disappeared and the chitin layer was thinner than in the control. The eggs treated with chitinases displayed large vacuoles in the chitin layer, and the vitelline layer was split and had lost its integrity. The major changes in the eggshell structures occurred by the combined effect of P. lilacinus protease and chitinases. The lipid layer was destroyed; the chitin layer hydrolyzed and the vitelline layer had lost integrity. The effect of P. lilacinus protease and chitinase enzymes on the hatching of M. javanica juveniles was also compared with a commercially available bacterial chitinase. The P. lilacinus protease and chitinase enzymes, either individually or in combination, reduced hatching of M. javanica juveniles whereas a commercial bacterial chitinase had an enhancing effect. Some juveniles hatched when the eggs were exposed to a fungal protease and chitinase mixture. We also established that P. lilacinus chitinases retained their activity in the presence of endogenous protease activity.     

Der Besatz von Beta-Rüubensaatgut rait Wurzelbranderregern und der Einfluß von Umweltfaktoren auf das Auftreten des samenbüurtigen Wurzelbrandes

The root knot nematode Meloidogyne incognita was controlled more effectively when P. lilacinus and G. mosseae were applied together in a pot experiment than either was applied alone. Inoculation of tomato plant with G. mosseae did not markedly increase the growth of plant infected with M. incognita. Inoculation of plant with G. mosseae and P. lilacinus together or alone resulted in a similar shoot and plant height. The highest root development was achieved when mycorrhizal plant were inoculated with P. lilacinus to combat root knot nematode. Inoculation of tomato plant with P. lilacinus suppressed galls/root system and eggs/egg masses, compared to seedling inoculated with M. incognita alone. The mycorrhizal colonization was not affected by inoculation of P. lilacinus.     

Impact of Paecilomyces lilacinus Inoculum Level and Application Time on Control of Meloidogyne incognita on Tomato

Microplot experiments were conducted to evaluate the effects of inoculum level and time of application of Paecilomyces lilacinus on the protection of tomato against MeIoidogyne incognita. The best protection against M. incognita was attained with 10 and 20 g of fungus-infested wheat kernels per microplot which resulted in a threefold and fourfold increase in tomato yield, respectively, compared with tomato plants treated with this nematode alone. Greatest protection against this pathogen was attained when P. lilacinus was delivered into soil 10 days before planting and again at planting. Yield was increased twofold compared with yield in nematode-alone plots and plots with M. incognita plus the fungus. Percentages of P. lilacinus-infected egg masses were greatest in plots treated at midseason or at midseason plus an early application, compared with plots treated with the fungus 10 days before planting and (or) at planting time.     

Effect of Plant Species on Persistence of Paecilomyces lilacinus Strain 251 in Soil and on Root Colonization by the Fungus

The effect of 12 plant species on the persistence of Paecilomyces lilacinus strain 251 in soil was investigated. After incorporating formulated conidia into non-sterile soil followed by transplanting different test plants, the population dynamic of the fungus was determined over 100 days. At termination of the experiment, the fungal population in the planted soil was compared to the density of P. lilacinus in the rhizosphere and the percent increase or decrease was calculated for each crop. In addition, the potential of P. lilacinus strain 251 to colonize roots endophytically was investigated. Comparison of the slopes describing the population dynamics of the fungus showed no significant differences between soil without plants and soil from the root zone of the majority of the test plants. Bean was the only plant species consistently exerting a negative effect on the persistence of P. lilacinus strain 251 in the soil. For the first time, P. lilacinus strain 251 was isolated in significant numbers from healthy root tissue of barley plants.     

Evaluation of Argemone mexicana for Control of Root-Infecting Fungi in Tomato

Argemone mexicana L. (Papaveraceae), a tropical annual weed, is known to be phytotoxic to many crop species. This study was designed to examine the possible impact of A. mexicana on root‐infecting fungi, changes in fungal community structure and the growth of tomato. A. mexicana decaying shoots in soil provided a marked decrease in the infectivity of Fusarium solani and Rhizoctonia solani but Macrophomina phaseolina remained unaffected. Plant height and shoot growth of tomato plants increased markedly though high concentration of A. mexicana (5% w/w) was deleterious to tomato plants. General species diversity of soil fungal communities increased in the amended soils over the controls and greater increase in diversity occurred at higher concentrations of decaying A. mexicana. Likewise, equitability and richness components of diversity increased in treatments compared to controls but declined with increasing sampling period. Aspergillus nidulans, Cephaliophora irregularis, Drechslera halodes, Paecilomyces lilacinus and Trichoderma viride were isolated exclusively from the amended soils. Aqueous extract of A. mexicana when applied in soil greatly suppressed all three of the above root‐infecting fungi, and at lower concentration actually enhanced plant growth. The influence of different levels of N‐fertilization with NH₄NO₃ on the modification of the effect of decaying A. mexicana on root‐infecting fungi was also investigated. N‐fertilization to some extent alleviated the phytotoxicity to tomato plants while suppressing the root‐infecting fungi. A. mexicana in conjunction with Pseudomonas aeruginosa, a plant growth‐promoting rhizobacterium, significantly suppressed root‐infecting fungi with concomitant increase in plant growth. Whereas P. aeruginosa was reisolated from the rhizosphere and inner root tissues of tomato, its population slightly declined in the amended soil but not to an extent that could reduce the biocontrol and growth promoting potential of the bacterium.