Measuring abundance,diversity and parasitic ability in two populations of the nematophagous fungus Pochonia chlamydosporia var. chlamydosporia

Abundance, genetic diversity and parasitic ability in the facultative nematode parasite Pochonia chlamydosporia var. chlamydosporia were compared in soils from two sites in Portugal under long-term tomato cultivation where root-knot nematodes (Meloidogyne sp.) were present. Fungal abundance assessed by selective agar or real-time quantitative PCR with specific primers was similar in both soils. PCR fingerprinting of isolates with ERIC primers indicated that the dominant P. c. var. chlamydosporia biotypes (profiles A and B) in both soils were very closely related, although a second biotype (profile C) was detected in one soil. When tomato plants infected with M. incognita were grown in the two soils, only profiles A and B were recovered from eggs. Primers based on polymorphisms in vcp1 demonstrated that isolates with profiles A and B were likely to prefer root-knot nematodes, whereas profile C preferred cyst nematodes. In the soil containing profiles A, B and C, egg parasitism by P. chlamydosporia was estimated at 1% using water agar plates with antibiotics but fewer than 0.2% of M. incognita eggs were shown to be infected with P. c. var. chlamydosporia when using species-specific β-tubulin-PCR primers. In contrast, the soil containing only profile B showed 22% egg parasitism on water agar plates and more than 2.5% of eggs were confirmed as P. c. var. chlamydosporia by species-specific β-tubulin-PCR primers. The results, which reveal limited diversity within the fungus at the two sites, are discussed in relation to biological control of plant-parasitic nematodes.     

Detection and Investigation of Soil Biological Activity against Meloidogyne incognita

Greenhouse experiments with two susceptible hosts of Meloidogyne incognita, a dwarf tomato and wheat, led to the identification of a soil in which the root-knot nematode population was reduced 5- to 16-fold compared to identical but pasteurized soil two months after infestation with 280 M. incognita J2/100 cm³ soil. This suppressive soil was subjected to various temperature, fumigation and dilution treatments, planted with tomato, and infested with 1,000 eggs of M. incognita/100 cm³ soil. Eight weeks after nematode infestation, distinct differences in nematode population densities were observed among the soil treatments, suggesting the suppressiveness had a biological nature. A fungal rRNA gene analysis (OFRG) performed on M. incognita egg masses collected at the end of the greenhouse experiments identified 11 fungal phylotypes, several of which exhibited associations with one or more of the nematode population density measurements (egg masses, eggs or J2). The phylotype containing rRNA genes with high sequence identity to Pochonia chlamydosporia exhibited the strongest negative associations. The negative correlation between the densities of the P. chlamydosporia genes and the nematodes was corroborated by an analysis using a P. chlamydosporia-selective qPCR assay.     

Interaction between Meloidogyne incognita and Pochonia chlamydosporia and their effects on the growth of Phaseolus vulgaris

An experiment was conducted to test the effect of different doses of 2, 4 and 8?g/2?kg of soil of Pochonia chlamydosporia against the root-knot nematode (Meloidogyne incognita) on Phaseolus vulgaris. It was observed that inoculation of plant with the nematode alone, and 15?days prior to fungal inoculation, reduced the plant growth when compared with the plant with fungal application followed by the nematode. Plant length, fresh and dry weight, chlorophyll, carotenoid, protein contents and nitrate reductase activity decreased in nematode-infested plants. Application of higher dose of 8?g/2?kg of soil of P. chlamydosporia increased all the plant growth parameters as well as biochemical parameters. Highest number of galls per root system was recorded on the plants infested with nematode but not treated with the fungus. However, application of fungus prior to nematode inoculation improved the plant growth and reduced the number of galls and the number of egg masses per root system.     

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.     

Effects on plant growth and root-knot nematode infection of an endophytic GFP transformant of the nematophagous fungus Pochonia chlamydosporia

Pochonia chlamydosporia (Pc123) is a fungal parasite of nematode eggs which can colonize endophytically barley and tomato roots. In this paper we use culturing as well as quantitative PCR (qPCR) methods and a stable GFP transformant (Pc123gfp) to analyze the endophytic behavior of the fungus in tomato roots. We found no differences between virulence/root colonization of Pc123 and Pc123gfp on root-knot nematode Meloidogyne javanica eggs and tomato seedlings respectively. Confocal microscopy of Pc123gfp infecting M. javanica eggs revealed details of the process such as penetration hyphae in the egg shell or appressoria and associated post infection hyphae previously unseen. Pc123gfp colonization of tomato roots was low close to the root cap, but increased with the distance to form a patchy hyphal network. Pc123gfp colonized epidermal and cortex tomato root cells and induced plant defenses (papillae). qPCR unlike culturing revealed reduction in fungus root colonization (total and endophytic) with plant development. Pc123gfp was found by qPCR less rhizosphere competent than Pc123. Endophytic colonization by Pc123gfp promoted growth of both roots and shoots of tomato plants vs. uninoculated (control) plants. Tomato roots endophytically colonized by Pc123gfp and inoculated with M. javanica juveniles developed galls and egg masses which were colonized by the fungus. Our results suggest that endophytic colonization of tomato roots by P. chlamydosporia may be relevant for promoting plant growth and perhaps affect managing of root-knot nematode infestations.     

Effect of plant growth promoting rhizobacteria,nematode parasitic fungi and root-nodule bacterium on root-knot nematodes Meloidogyne javanica and growth of chickpea

Effects of plant growth promoting rhizobacteria (Pseudomonas putida MTCC No. 3604 and Pseudomonas alcaligenes MTCC No. 493) and parasitic fungi (Pochonia chlamydosporia KIA and Paecilomyces lilacinus KIA) were studied, alone and together with Rhizobium sp. (charcoal commercial culture) on the growth of chickpea and multiplication of Meloidogyne javanica. Individually, P. putida 3604, P. alcaligenes 493 and Rhizobium caused a significant increase in the growth of chickpea in both nematode inoculated and uninoculated plants. Inoculation of Rhizobium with a parasitic fungus or with plant growth promoting rhizobaterium caused a greater increase in the growth of plants inoculated with nematodes than caused by either of them singly. Individually, P. lilacinus KIA caused a greater increase in the growth of nematode inoculated plants than caused by P. putida 3604 or P. alcaligenes 493. P. lilacinus KIA caused a greater reduction in galling and nematode multiplication followed by P. chlamydosporia KIA, P. putida 3604 and P. alcaligenes 493. Combined use of P. lilacinus KIA with Rhizobium was better in reducing galling and nematode multiplication than any other treatment. P. putida 3604 caused a greater colonization of root than P. alcaligenes 493 while P. lilacinus KIA was isolated from more nematodes than P. chlamydosporia KIA.     

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.     

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.     

Identification and molecular characterisation of new Mexican isolates of Pochonia chlamydosporia for the management of Meloidogyne spp.

New Mexican isolates of the nematophagous fungus Pochonia chlamydosporia were obtained from nematode infested fields in the vegetable growing area of Tepeaca Valley, Puebla State, Mexico. Based on macro and microscopic morphology, seven ‘putative’ P. chlamydosporia isolates were selected and the DNA extracted for polymerase chain reaction (PCR). Three new isolates of P. chlamydosporia were identified: Pcp2, Pcp21 and Pcp31. The amplification reaction of the internal transcribed spacer (ITS) region revealed a 650 bp amplicon which was used in a maximum likelihood phylogenetic inference analysis. Three groups were recovered in the tree topology, supported by a > 90% bootstrap value. Nucleotide identity values were > 83.6% between the test sequences and the reference sequence. In addition, using specific primers for two existing varieties of P. chlamydosporia, restriction fragment length polymorphism on the ITS products in conjunction with the phylogenetic inferences and the molecular test for detection of P. chlamydosporia vcp1 gene, it was found that all three isolates belong to a new variety which we have named P. chlamydosporia var. mexicana. We compared the chlamydospore production rate, rhizosphere colonisation and egg parasitism percentages of the three native isolates in Meloidogyne spp. with a reference isolate (Pc10). Native isolates produced > 1×10⁶ chlamydospores/50 g of substrate (of which more than 80% were viable), colonised > 80% of the rhizosphere, and parasitised > 60% of Meloidogyne incognita and Meloidogyne arenaria eggs. Meloidogyne hapla egg parasitism was < 60%. Isolates Pcp2 and Pcp21 were identified as potential biological control agents of Meloidogyne spp. to be tested further in greenhouse and field tests.     

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.     

Relationship between saprotrophic growth in soil of different biotypes of Pochonia chlamydosporia and the infection of nematode eggs

The ecology of Pochonia chlamydosporia in soil and its interaction with both plant and nematode hosts are important for the successful exploitation of the fungus as a biological control agent. Differences in saprotrophism and parasitism were assessed for biotypes of P. chlamydosporia, which had originated from the eggs of cyst or root‐knot nematodes. Colonisation in soils of different textures (compost, sandy loam and loamy sand) measured by the numbers of colony‐forming units, differed greatly. Most biotypes were more abundant in sterilised soil of the different textures compared with non‐sterilised soils. The proportion of nematode eggs parasitised in a baiting technique demonstrated that biotypes had host preferences. Those biotypes that originated from root‐knot nematodes (RKN‐biotypes) infected significantly more Meloidogyne hapla eggs than Globodera pallida eggs, whereas biotypes from cyst nematodes (CN‐biotypes) parasitised more G. pallida eggs than M. hapla eggs. Differences in virulence between biotypes in an in vitro assay in which the fungi were placed directly onto the egg masses of M. hapla and those differences observed in the baiting technique showed similar trends. There was a negative linear correlation between the growth of the eight biotypes in soil and the proportion of eggs they infected in compatible interactions (i.e. fungal biotype originated from the same nematode genus as the target eggs). Those biotypes that infected most nematode eggs colonised soil the least extensively, suggesting that virulence may have a fitness cost. However, the relationship between saprotrophic growth and virulence is complex. The relative abundance of the different biotypes in soil in Petri dish assays was similar to that under glasshouse conditions using potato but not tomato as the plant host. Chlamydospores of some biotypes applied to soil significantly reduced (>50%) the population densities of M. hapla on tomato and of G. pallida on potato plants. Some biotypes that were both effective and virulent are good candidates for biological control of specific nematode pests. Data presented here and elsewhere indicate that RKN‐biotypes have different host preferences to CN‐biotypes; the specific primers based on the vcp1 gene from P. chlamydosporia rapidly confirmed the host origin of seven of the eight biotypes.     

Studies on the disease complex incidence by Meloidogyne incognita and Fusarium solani on Poi,Basella rubra

A survey of Poi crop in Ghaziabad (UP) exhibited a disease complex incidence by Meloidogyne incognita and Fusarium solani causing synergistic effect on the host. Paecilomyces lilacinus was found from the egg masses of M. incognita and Aspergillus niger and Aspergillus terreus from the rhizosphere of root-knot infected Poi crop. Paecilomyces lilacinus parasitised the eggs to a greater extent. The level of parasitism was highest (65%) by P. lilacinus while Aspergillus spp. did not colonise the eggs. Fusarium solani which in the present investigation has been established to be pathogenic to Poi plant.     

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.     

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.     

Low-level herbivory by root-knot nematodes (Meloidogyne incognita) modifies root hair morphology and rhizodeposition in host plants (Hordeum vulgare)

Low amounts of root infestation by plant parasitic nematodes are suggested to increase nutrient supply and in turn enhance microbial activity and net mineralization rate in the rhizosphere. These effects are generally related to “leakage” of plant-derived metabolites from damaged roots. Besides leakage, the present study examines other nematode–host interactions such as alterations in root exudation and morphology, which were almost not considered yet. This includes undamaged root parts in order to assess systemic plant response. The root-knot nematode Meloidogyne incognita (Kofoid and White 1919; Chitwood 1949) and barley (Hordeum vulgare L. cv. Europa) was used as model system. Host plants were grown in mini-rhizotrons inoculated with 0, 2,000, 4,000 or 8,000 M. incognita for 4 weeks. Root morphology, rhizodeposition (sugars, carboxylates, amino acids), and rhizosphere microbial communities (PLFAs) were assessed. In treatments with 4,000 nematodes, shoot biomass, total N and P content increased by the end of the experiment. Generally, an enhanced release of plant metabolites (sugars, carboxylates, amino acids) from the apical root zone occurred 1 week after inoculation with 4,000 and 8,000 M. incognita, indicating root leakage. Low levels of root herbivory stimulated root hair elongation in both infected and uninfected roots. These systemic changes in root morphology likely contributed to the increased sugar exudation in uninfected roots in all nematode treatments at 3 weeks after inoculation. Root-knots formed a separate microhabitat within the root-system. They were characterised by decreased rhizodeposition and increased fungal to bacterial ratio in the adhering rhizosphere soil. The present study provides the first evidence that, apart from leakage, nematode root herbivory at background levels induces local and systemic effects on root morphology and exudation, which in turn may affect plant performance.     

Biocontrol Efficacy Among Strains of Pochonia chlamydosporia Obtained from a Root-Knot Nematode Suppressive Soil

Three Pochonia chlamydosporia var. chlamydosporia strains were isolated from a Meloidogyne incognita-suppressive soil, and then genetically characterized with multiple Pochonia-selective typing methods based on analysis of ß-tubulin, rRNA internal transcribed spacer (ITS), rRNA small subunit (SSU), and enterobacterial repetitive intergenic consensus (ERIC) PCR. All strains exhibited different patterns with the ERIC analysis. Strains 1 and 4 were similar with PCR analysis of ß-tubulin and ITS. The strains'' potential as biological control agents against root-knot nematodes were examined in greenhouse trials. All three P. chlamydosporia strains significantly reduced the numbers of nematode egg masses. When chlamydospores were used as inoculum, strain 4 reduced egg numbers on tomato roots by almost 50%, and showed effects on the numbers of J2 and on nematode-caused root-galling. A newly developed SSU-based PCR analysis differentiated strain 4 from the others, and could therefore potentially be used as a screening tool for identifying other effective biocontrol strains of P. chlamydosporia var. chlamydosporia.     

根际效应对大豆田土壤线虫群落组成及多样性的影响

根际作为重要的环境界面是植物与环境之间物质能量交换的场所,关于根际效应的研究已成为土壤生态学的新兴热点领域,然而有关大豆根际效应对土壤动物多样性影响的研究报道并不多见。在三江平原选择连续耕作15a的大豆田,对大豆根际区与非根际区土壤线虫群落结构组成进行了对比分析。结果表明:大豆根际区土壤线虫总数、辛普森多样性指数(Dom)显著高于非根际区,根际区的物种数(S)、物种丰富度指数(SR)显著低于非根际区。说明大豆根际效应增加土壤线虫的丰度,但降低了线虫群落结构的复杂性。大豆根际区植物寄生线虫(PP)、食真菌线虫(FF)和食细菌线虫(BF)数量显著高于非根际区,而PP类群的比例在根际区却显著低于非根际区。这一研究结果表明食微线虫(FF和BF)类群在大豆根际区的比例增加更显著。食真菌与食细菌线虫数量比值(F/B)指示大豆根际区细菌生物量相对高于真菌生物量。研究结果丰富了农田土壤线虫多样性的研究内容,并为我国东北大豆田线虫病害的防治及定制相应的农业管理措施提供参考。     

Persistence of the nematophagous fungus Paecilomyces lilacinus strain 251 in soil under controlled conditions

The persistence of the nematophagous fungus Paecilomyces lilacinus Samson strain 251 (PL251) and the effect of application rate, substrate type, as well as the presence of the nematode host on its dynamics after application to the soil were investigated under controlled conditions. In all experiments, increase of P. lilacinus colony forming units after application was not found. In contrast, a gradual decline in fungal densities over time was observed. Application rate had no significant effect on the dynamics of the fungal population. Likewise, P. lilacinus density decline in soil was not significantly affected by the presence of the nematode host. Substrate type had a significant effect on P. lilacinus persistence in soil. The fungal agent persisted longer in silty loam and clay soil, with reduced persistence when sand was added to field soil. Conversely, when organic substrate was added to pure sand, persistence was significantly increased. Although persistence of fungal biocontrol agents in soil depends on various biotic and abiotic conditions, baseline data on persistence such as those reported in this study are helpful for biocontrol and environmental risk assessment and merit further study.     

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.