Egg and Female Parasitic Fungi 4231 Introduction

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In contrast with the numerous migrating nematodes, some plant-pathogenic nematodes spend the majority of their life cycle inside plant roots or on their surface in cysts and/or in root knots. These sedentary stages persist in the soil and serve as a selective substratum for fungal colonization by egg parasites. Many opportunistic soil fungi have been isolated from the eggs, cysts and sedentary females that lay their eggs in gelatinous matrices, such as Meloidogyne spp. and Tylenchulus semipenetrans [2, 11, 20, 157-159]. Generally, egg and cyst parasitizing fungi are more numerous than those infecting females [160]. This group of fungi uses appressoria or zoospores to infect their hosts [16]. The parasites of egg and sedentary stages have attracted more attention because of their high potential in biological control of economically important nematodes. These fungi that can saprotrophically survive well in rhizo-sphere, are relatively easy to mass-culture and are more effective in infecting because their host is sessile (eggs, developing juveniles and females).

Among all nematode parasitizing fungi, comparatively few have been considered as promising biocontrol agents [14], and of these the most frequently isolated fungi are Pochonia chlamydosporia and Paecilomyces lilacinus [14, 161-164]. Species of Pochonia, Paecilomyces, Haptocillium, and Hirsutella are among the most favorable biocontrol agents against plant-parasitic nematodes [3, 6, 18, 113, 166-169]. Taxonomy

Recently, the egg-parasitic fungi previously accommodated under the genus Verticillium were transferred to the genus Pochonia according to both morphological and molecular characters [170, 171]. Pochonia species mostly produce dictyo-chlamydospores or at least some irregularly swollen hyphae. The production of dictyochlamydospores was mostly used to characterize Diheterospora, but this is an unreliable character for distinguishing species of this genus, because they are absent or scanty in some species, while similar structures also occur in species of Rotiferophthora and Haptocillium [171]. Their species can be more or less easily distinguished on the basis of conidial shape and the position and abundance of dictyochlamydospores [170, 171]. The teleomorphs of Pochonia species are located within Metacordyceps [172]. Although all Pochonia species could parasitize Meloidogyne javanica eggs [173] and considered as the best egg colonizers, but other species like Paecilomyces lilacinus and Lecanicillium lecanii are also effective in egg parasitization [16].

In addition to the mentioned facultative parasites in the Hyphomycotina, other fungi belonging to Oomycota (Nematophthora gynophila and an undescribed lagenidiaceous fungus) and Chytridiomycota (Catenaria auxiliaris) are also reported as obligate parasites of cyst nematode females [6]. Ecology

Paecilomyces lilacinus is abundant and active in subtropical and tropical areas [11], while Lecanicillium is mainly found in tropical areas [171]. Lecanicillium is reported from W. Indies, Dominican Republic, Peru, Jamaica, USA, Sri Lanka, Indonesia, Iran and Turkey [174-176]. Nematophthora gynophila is prevalent in soils of northern Europe infested with cereal cyst nematode and usually occurs together with P. chlamydosporia, and both are involved in declining of this pest species [2]. Pochonia chlamydosporia, P. bulbillosa, Paecilomyces marquandii, P. lilacinus, and P. carneus were isolated from Ascaris eggs buried in soils in the Czech Republic, Pakistan, Afghanistan, and Cuba. Pochonia spp. and P. lilacinus rapidly infected and killed the eggs [177]. Pochonia chlamydosporia is one of the most cosmopolitan species, but its Metacordyceps teleomorph is so far known only from slug eggs in the tropics [178]. Pochonia chlamydosporia is the major egg pathogen of Heterodera species in all European and American countries examined [3, 113, 179]. The species is also found as an efficient parasite of Meloidogyne root-knot nema-todes [164, 180-182].

Pochonia suchlasporia was a rather common fungus in central and northern Europe [20] especially on Heterodera cysts in Denmark, Sweden and the Netherlands [183-185], while P. chlamydosporia is more restricted to young cysts in these countries [171].

Some species, such as Paecilomyces lilacinus and Pochonia spp. are presumably not influenced by antimicrobial activity of the matrices produced by root-knot [187] and cyst nematodes [2]. These fungi are more abundant on galled roots infected by Meloidogyne spp. than in the rhizospheres of healthy roots [188], and their isolates have been collected from a broad range of cyst and root-knot nematodes with a worldwide distribution [2]. Two distinct barriers impede the infection of nematode eggs by fungi, the eggshell and the cuticle of the second stage larvae within the egg [17], therefore, immature eggs are more prone to parasitism than those containing larvae (Fig. 4.4). Pochonia chlamydosporia colonizes dead eggs of Heterodera more efficiently than live ones, with a trophic favorite of young stages, before the embryo development is completed [189].

Many experiments illustrated that egg-parasitic fungi are preferably inhabited at rhizosphere [15, 190]. Plant species influence the growth of P. chlamydosporia [190], and nematode parasitism may help support the long period maintenance of the fungus in soil [2, 191]; although the fungus is more effective when applied on poor hosts for the nematode, than when applied on fully susceptible crops.

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Fig. 4.4 Parasitized eggs of M. javanica infected by Pochonia chlamydosporia var. catenulata (a) condiophore on an egg, (b and c) dictyo-chlamydospore associate with an immature and mature infected egg [173]

Inoculation of the nematode poor host plants prior to a susceptible host could assist the fungus to construct a high level of population density that could manage the nematode efficiently [20]. It seems that the fungus could not provide a sufficient nematode control by itself and must be integrated with other managing measures [140, 181, 192]. All egg and female parasites grow willingly on artificial media and some produce resting spores which serve as a survival stage in soil [2].

Different isolates of Pochonia spp. differ in virulence [15], ability to colonize the root epidermis and cortex [66], and dictyochlamydospore production [171], while all of these characters are considered important for the use of the fungus as a biocontrol agent.

Dictyochlamydospores are usually used as an inoculum to introduce and to establish the fungus in the soil and rhizosphere. There are some biological (dilution plating on a selective media) and molecular (PCR, real-time PCR, and RFLP) methods which are developed to screen the presence, abundance and activity of the fungus in the soil, rhizosphere and nematode egg masses [193]. Isolates that obtained from cyst nematodes have greater ability to parasitize the cyst nematode eggs than isolates recovered from root-knot nematodes, and therefore it is suggested that the fungus has host preference [11, 173].

We know a little about the factors that cause switching trophic state from sapro-trophs to parasites. In contrast with more abundance of P. chlamydosporia in organic soils, its antagonistic activity may be no greater than in a mineral soil [11]. The fungus can be formulated and introduced as fungal hyphae and conidia, but dictyochlamydospores are the most popular form of inoculum. Single application of 5,000 dictyochlamydospores per gram soil of vegetable crops in tropical soils provided sufficient control of root-knot nematodes but in Europe the results have been less satisfactory [11].

The ecology of P. chlamydosporia has been the subject of many studies [18, 164, 180, 181, 183, 184, 186, 188, 194, 195]. Among all species of Pochonia, P. chlamydosporia var. chlamydosporia has been studied extensively as a potential biocontrol agent against nematodes [15, 181]. Its teleomorph, Metacordyceps chlamydosporia, has been found on slug eggs in tropical countries [20]. There are also some reports on the ability of other members of this genus like P. c. var. catenulata [193, 196], two varieties of P. suchlosporia [183, 186], P. rubescens [197, 198], P. bulbillosa and P. globispora [173] parasitizing nematode eggs. Managing of cyst nematodes and root-knot nematodes by this fungus in greenhouse and microplot trials has been repeatedly reported [161-163, 173, 199, 200]. Pochonia chlamydosporia is also ovicidal to the large roundworm, Ascaris lumbricoides [201] and slug eggs [202, 203].

In peanut fields, Meloidogyne arenaria was more frequently parasitized by P. chlamydosporia than Heterodera glycines [163]. In contrast with many successful trails of the potential application of Pochonia chlamydosporia against plant parasitic nematodes [3, 161, 164, 169, 181, 204], its first use of conidial suspensions was failed [205].

Lopez-Llorca and Duncan [198] illustrated the colonization of Heterodera avenae by species of Pochonia using SEM. Pochonia chlamydosporia can be effectively integrated with the nematicide Aldicarb. The nematicide mostly prevents initial nematode injury, while the fungus part causes a long-term protection. Aldicarb did not influence the activity of the fungus and resulted in a better control of M. hapla than treatment with Aldicarb or P. chlamydosporia alone [200].

Using an isolate of P. c. var. catenulata in combination with crop rotation notably reduced nematode populations in soil following a tomato crop [193]. The fungus can also be introduced in combination with an arbuscular mycorrhizal symbiont, Glomus desertorum. Their combination added to tomato nursery seedlings resulted in more efficient control. In that experiment 68% of eggs parasitized, while P. chlamydosporia alone could parasitize 52% of eggs [206]. Application of P. chlamydosporia together with chopped leaves of Azadirachta indica (neem) acted synergistically in reducing both gall index and nematode population in tomato in pot experiments [207]. Applying the combination of P. chlamydosporia, Trichoderma harzianum, and Glomus mosseae significantly controlled Heterodera cajani on pigeon pea [208]. The consistency of such approaches needs extensive assessment.

The biology of P. chlamydosporia and its potential for biological control of cyst and root-knot nematodes has been reviewed [181, 192]. The fungus proliferates in calcareous loams and organic soil in England and could survive at least 3 month after application, however, different isolates varied significantly in their survival ability and also in proliferation in different soils [11, 166, 195]. The fungus prefers peaty sand soil rather than loamy sand or sand in tomato plots infected with M. incognita; however in sandy loam microplots a 90% control of M. hapla could be achieved, only if the temperature did not exceed 25°C [200]. Its optimal pH for growth was 6, but some could grow even at pH 3 [120]. Control of H. schachtii was directly related to the quantity of young infected females but not to the number of colonized cysts. Effective control will achieve only if few egg produced and many of them were colonized [167]. In tomato soils, fluctuations in population densities of M. incognita and P. chlamydosporia followed each other, and supplemented soil with Meloidogyne species cause a population increscent of the fungus [20]. The fungus can naturally decline the nematode populations [209] and partial sterilization of the soil with 38% formaldehyde destroyed the nematode-decline effect according to killing fungus population [49].

It was observed that addition of wheat bran to alginate pellets was essential for the successful and consistent establishment of the fungus [195], while de Leij and Kerry [161] had found that adding dictyochlamydospores and hyphal fragments without any extra food base would result in the best establishment. Supplemented the inoculum with an energy producing base could enhance competition from the residual microflora that may cause an adverse effect on survival and multiplication of Pochonia [210]. In the other hand, it seems that the fungus (especially hyphae and conidia inocula) essentially needs an energy source for its establishment in mineral soil [211]. The comprehension of the tritrophic system is important for a successful application [212].

Approximately 103-104 CFU of the fungus per gram soil usually suppresses the cyst nematodes, and dictyochlamydospores are regarded as more efficient than alginate-bran pellets [213]. Up to 43% of egg masses of M. hapla were parasitized when 5,000 dictyochlamydospores were added to each cm3 of soil, but no effect was observed on lettuce weight, root galling, or egg production [122]. When the population of nematode is high, a successful biocontrol could not be expected [214]. The fungus could produce about 5 x 106 dictyochlamydospores per gram of sand-barely bran medium mixture [49]. On the condition that extra nutrients were added, fungus strongly proliferated in soil [214]. Some isolates of P. chlamydosporia reproduce in the rhizosphere of appropriate host plants without any unfavorable effects on the plant [161, 212, 213, 215].

For a successful exploitation of P. chlamydosporia as an efficient biological control agent, not only a detailed information of the molecular mechanism involved in infection of the nematodes is required, but also of ecological concern is the population dynamics of the fungus in the rhizosphere [15]. Devising a biological control strategy, it is vital to understand the dynamics of P. chlamydosporia in relation to the nematode populations; however, interpreting the basic information of such an approach is difficult due to difficulties in quantifying the fungus in the rhizosphere and to the lack of a simple relationship linking fungal abundance to its activity [17]. Quantification of filamentous fungi is not easy since they are not composed of single, simple-to-quantify units of nearly the same size. Like other fungi, P. chlamydosporia has several life stages that comprises of multicellular hyphae and dictyochla-mydospores mixed together with unicellular conidia. Consequently, understanding a robust association of fungal biomass with nematode numbers, which practically relates to nematode infection and control, is problematic because any component of fungus quantified could result from any of the life stages, even the resting stages which not certainly contribute in nematode colonization at the time of estimation [17].

Several methodologies are in hand for such examinations, including selective plating, immunological and PCR-based techniques [216]. The dictyochlamydospores could be extracted and enumerated from soil [217]. Media for the selective isolation and quantification of P. chlamydosporia were devised by Gaspard et al. [188, 194] (a chitin-rose bengal agar with 50 mg/L benomyl); de Leij and Kerry [161] and Kerry et al. [195] recommend cornmeal agar (Oxoid) with 37.5 mg carbendazim, 37.5 mg thiabendazole, 75 mg rose bengal, 17.5 mg NaCl, 3 mL Triton X-100, and antibacterial antibiotics; and Moosavi et al. [173] used Shrimp-Agar medium with 3 g shrimp shell powder, 17 g agar, 37.5 mg carbendazim, 37.5 mg thiabendazole, 17.5 mg NaCl, 3 mL Triton X-100, and 200 ppm each streptomycin sulphate and penicillin.

Kerry and Crump [218] described a quantification method for diseased eggs of Heterodera species. Egg parasites of cyst nematodes could also be quantified by a standard technique in which the cysts were extracted from soil and then were crushed, afterward their contents were reincorporated into the original soil sample and colonization activity on newly produced eggs is then assessed [219]. Several methods were devised to quantify the ability of the fungus in colonizing the plant rhizosphere in sterile and unsterile soil [190]. Specific primers for the P-tubulin gene of P. chlamydosporia are developed to detect the fungus on infected plant roots [216, 220]. It is documented that the most precise explanation of fungal dynamics in the soil could only be achieved by combining culture- and PCR-based techniques together rather than using either method alone [221].

Pochonia suchlasporia was particularly successful in colonizing eggs and exhibited more chitinase and protease activities [183, 184]. The fungus secretes several extracellular enzymes (especially protease, chitinase and collagenase) that serve as virulence factors involving in pathogenicity [222]. Tikhonov et al. [46] demonstrated that P. suchlasporia always secretes higher volume of enzymes compared with P. chlamydosporia. Further production of enzymes may be the key factor for this species that must be considered, while scanty dictyochlamydospore production [171] can be a disadvantage for this species which affects its dispersal and survival in the soil. Comparing with P. chlamydosporia, this fungus has also a lower minimum and optimum temperature for growth [20] that could limit its commercial application in many countries with warm climate (including the Mediterranean). As optimum growth temperature for this variety is measured (18-21°C) [171] this fungus can be a good candidate for temperate and cool regions. The most effective isolate of P. chlamydosporia (V. chlamydosporium) tested by Irving and Kerry [189] was also infectious at 5°C and should probably be identified as P. suchlasporia.

The ex-type strain of P. rubescens was isolated from eggs of H. avenae [178, 197, 198]. The fungus showed optimal growth at pH 6, but generates red pigments on acidic media [120] that when was extracted in chloroform/methanol had nematicidal effect on potato cyst nematode, Globodera rostochiensis [223]. This fungus parasitized eggs of Heterodera and Globodera species in vitro, and as demonstrated in TEM photographs, developing appressoria and penetration hyphae with an interior infection bulb [224].

Describing P. globispora in the genus Pochonia, Zare and Gams [225] have anticipated the potential of this species as a possible biocontrol agent of nematodes. Pochonia bulbilosa is usually recovered from forest soils [171], but apart from its isolation as an ovicidal species from Ascaris eggs in Pakistan and Afghanistan [177], its association with nematodes had not been sufficiently known [20]. Moosavi et al. [173] reported effectiveness of the last two species in colonizing M. javanica eggs.

Pochonia gonioides was originally observed on a species of Bunonema, but the mode of entry into the nematode could not be established [226]. Recently only two isolates of this species have been available, and these were originally not directly associated with nematodes [20].

Paecilomyces lilacinus is another facultative parasite that has been employed as a biological agent for the control of plant-parasitic nematodes. The fungus potential for human pathogenicity (ocular and cutaneous infections, onychomycosis, sinusitis, and deep infections in immunocompromized patients) [227, 228] seems to preclude its practical application, but some genetic differences were found between human-pathogenic isolates and the nematode parasites [20]. Paecilomyces lilacinus and the similar P. marquandii were isolated from eggs of Ascaris lumbricoides exposed in soils in the Czech Republic, Pakistan, and Cuba [177], and both fungi also penetrated, colonized and killed eggs of Toxocara canis, the canine roundworm [229]. Therefore P. lilacinus might also be applied as a biological control agent against animal helminths in vivo [20].

Paecilomyces Lilacinus
Fig. 4.5 Paecilomyces lilacinus conidiophores arising from an infected M. javanica egg

Paecilomyces lilacinus has a broad geographical distribution and was first observed in association with nematode eggs [230], and like P. chlamydosporia, it is principally regarded as an egg parasite (Fig. 4.5). Eggs of Meloidogyne incognita and Globoderapallida were efficiently colonized in Peru [231]. List of the sensitive nematode species to P. lilacinus were named in an extensive review [5]. Early examinations using P. lilacinus as a biocontrol agent were encouraging [5]; however, isolates known to be parasitic on nematode eggs and present at high population levels were unable to control root-knot nematodes [165, 232]. Many factors such as ecological components in connection to the establishment ability of the fungus in soil [232] and genetic factors important in determining levels of pathogenicity [158, 233] can be involved in this inconsistency.

Pathogenicity of different isolates of P. lilacinus to nematodes varies greatly [3]. Their pathogenicity was somewhat correlated with their UV resistance, that can also similarly be seen in grouping made by random amplified polymorphic DNA (RAPD) [234]. There is an inconsistency between greenhouse assays and those that were conducted in the field [204]. Paecilomyces lilacinus have also been used joined with organic resources like oil cake, leaf residues and seeds [14, 235]; but as usual, reliable control of nematodes has been difficult to achieve. An attempt was done to select low cost substrate for spore production of a nematicide strain of P. lilacinus. Coffee husks, cassava bagasse, and defatted soybean cake were utilized as substrates, and sugarcane bagasse was used as support. The products obtained by solid-state fermentation were tested for their nematicide activity against M. incognita in pot experiments containing Coleus as host plant. After 2 month the best results were achieved with defatted soybean cake, which showed almost 100% reduction in the number of nematodes, while the reduction with coffee husk was 80% and with cassava bagasse was about 60% [236]. The use of oxamyl 2 weeks before and during transplanting gave similar results to the commercial product containing P. lilacinus but superior to soil solarization [237].

The fungus has the ability to produce antibiotics (leucinostatin and lilacin) and chitinolytic enzymes [182]. Production of a serine protease which serves as a crucial component in disintegration of the eggshell is also documented [42, 238]. Decrease in Rotylenchulus reniformis population in tomato in India was accompanied with P. lilacinus population increment, which causes the level of control comparable to that by carbofuran [239]. In some Meloidogyne-suppressive soils in California, it seemed that P. lilacinus play an unimportant role in managing the nematode population [194]. Unlike positive correlation between population densities of P. lilacinus and P. chlamydosporia, no correlation was seen with Meloidogyne incognita [188]. Adding 10 or 20 g of fungus-colonized wheat kernels per a 76 cm diam microplot at planting time (even better with an additional treatment 10 days before planting) gave good protection against M. incognita and increased tomato yield significantly [240, 241]. Application of P. lilacinus in potato fields of Peru provided a lower galling index due to M. incognita than nematicide treatments [5], and the introduced fungus sufficiently established with a single application [242]. Different selective media have been devised for monitoring P. lilacinus by Cabanillas and Barker [241] (PDA with dichloran and oxgall together with antibacterial antibiotics) and Gaspard et al. [194] (chitin-rose bengal agar with 50 mg/L iprodione).

Economically production and formulation of filamentous fungal control agents remains problematic [243]; however, recent progresses in technology have made it possible to produce extremely concentrated formulations that can easily and successfully be used on a field scale [244-247]. Paecilomyces lilacinus strain 252 was developed as a commercial product in Germany (BioAct® WG) and South Africa (Pl Plus®) for cyst and root-knot nematode management. They are applied as dispersible granules for application in water [11, 247, 248].

Species of Lecanicillium are mostly entomogenous or fungicolous [171]. Lecanicillium psalliotae (once found in a cyst of Globodera rostochiensis) and "Verticillium" leptobactrum (mainly in Heterodera eggs) were occasionally isolated from nematodes [185]. Verticillium lecanii (= L. muscarium) was also rarely observed as parasites of cysts and eggs of Heterodera and Meloidogyne species [160, 180]. An insect isolate of "Verticillium lecanii" was examined for its in vitro controlling ability of Globodera pallida. After 2 months, eight isolates (most of them probably L. muscarium and one L. longisporum) out of 14 isolates could sufficiently colonize the eggs [249]. When L. lecanii was added to monoxenic soybean cyst nematode cultures, it successfully colonized the cyst and female; and reproduced in gelatinous matrix but no egg penetration was observed. Hence its antagonistic activity against soybean cyst nematode was attributed to chemical secretion [250]. Lecanicillium lecanii appears to be specific to soft scale insects, Coccidae or Lecanidae [171]. Mode of Action

The eggshell of nematodes is composed of three distinct layers and mostly consists of protein and chitin, which organized in a microfibrillar and amorphous structure [251]. These layers are an outer vitelline layer, a chitin layer and an inner lipoprotein layer [252]. Penetration to the eggshell of nematode occurs from an appressorium, a specialized penetration peg or lateral branches of mycelium [16]. Chitinases and proteases play an important role during eggshell penetration, and lead to disintegration of eggshell layers [253, 254]. The fungi which can produce more extracellular enzymes (especially protease, chitinase and collagenase) are considered much more effective in infection of nematode eggs [222], and it is demonstrated that fungi differ in their ability to degrade nematode eggshells, and infection process can be affected by the nematode host [238, 254]. Maybe the emanated signals from the egg influence fungal growth and development, and penetration of the eggshell [255].

The infection of nematodes and their eggs by various nematophagous fungi follows a similar, general pattern [16]. Pochonia chlamydosporia is regarded as a parasite of females and eggs of cyst and root-knot nematodes, and develops branched mycelial networks that form appressoria on the eggshell [11, 37, 164, 198].

Contact of the hyphae with the eggshell is the first step in penetrating nematode eggs by P. rubescens, followed by developing an appressorium covered with an extracellular material or adhesive. The extracellular material contains a protease (P32) that can be immunologically detected [16]. Deducing from labeling of the adhesive on the appressoria of P. chlamydosporia and P. rubescens with the lectin Concanavalin A, a glycoprotein nature with mannose/glucose moieties is suggested for that sticky material [256]. The fungus penetrates the nematode eggshell from the appressorium by means of both mechanical and enzymatic components. As the nematode eggshell mainly contains chitin and proteins [252], proteases, chitinases and lipases play an important role during eggshell penetration; however their penetration involvement have not as yet been examined to the same extent and detail [46, 197, 257]. Eleven isolates of Pochonia chlamydosporia that were kept in Rothamsted Research Station culture collection were selected and their ability in producing chitinases, esterases, lipases and serine protease (VCP1) were quantified and compared. The isolates were chosen so that they had different hosts, substrata and geographical origins. The results demonstrated that significant differences in enzyme production could be seen between different isolates, time of growth and the amounts of enzymes produced. No significant relationship were observed between trophic phase (parasitic or saprobic) and enzyme activities of the isolates, suggesting that switch in trophic phase is more complex and depended on several factors [258]. Pochonia chlamydosporia can degrade chitosan, an antifungal compound that severely affects plant pathogenic fungi, but not nematophagous and entomopathogenic fungi. It is demonstrated that the most abundant extracellular secreted proteins of P. chlamydosporia grown with chitosan as main carbon and nitrogen sources, involve in carbohydrate or protein degradation and egg penetration [259]. An endochitinase gene (pcchi44) [47] and a new serine carboxypeptidase (SCP1) genes [45] were isolated, identified and cloned from P. chlamydosporia.

Serine proteases were purified and characterized from P. rubescens [197], and P. suchlasporia [224]. Involvement of the enzyme in pathogenicity was suggested by its immunolocalization in appressoria of the fungus [38].

Pochonia chlamydosporia secretes the VCP1 protease that involves in hydrolyzing eggshell proteins of Meloidogyne species but not those of Globodera [254]. Thickness of the egg shells of Globodera are approximately twice as those of Meloidogyne which cause more resistance to disintegration [224]. A chymoelastase-like protease is also produced by P. chlamydosporia which has the ability of hydro-lysing host nematode proteins in situ [40]. Subtilisin-like proteases are the most important classes of extracellular enzymes that different isolates could have up to four isoforms of them. The enzymes decompose the proteins of their nematode hosts and are very important in fungal pathogenicity [238]. The similarity of the enzyme with that secreted by Metarhizium anisopliae is demonstrated [41].

It is also demonstrated that a serine protease and chitinases that are effective in degrading the eggshell, and a nematotoxin, phomalactone, which secreted by P. chlamydosporia enhance the pathogenicity [11, 254]. Further studies using Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR) revealed that different isolates of P. chlamydosporia produce a range of different proteases, and that the difference in the enzymes perhaps relates to the different ecological niches occupied by each fungus [238, 260].

ERIC-PCR generated data using in phylogenetic analysis illustrated that the different isolates of the fungus were related to its host from which the isolate had been obtained [253]. Comparison of the similarity of amino acid sequences between proteases from different nematophagous fungi showed a high level of conservation, with only minor insertions and deletions [261]. Minor variation in amino acid sequence may influence substrate utilization and host preference [41] that has been documented in VCP1 proteases from different isolates of P. chlamydosporia. Substitution of an alanine by a glycine in the S3 substrate-binding region of VCP1 confers enzymatic activity against eggshells of Meloidogyne [253].

Paecilomyces lilacinus is a well-studied antagonist of some nematodes like Radopholus similis and Tylenchulus semipenetrans, but most research has carried out on the infection of Meloidogyne spp. and Globodera rostochiensis eggs [11]. This fungus has been extensively evaluated for reducing nematode damage to a range of crops and its application usually caused a significant nematode control.

Paecilomyces lilacinus secrete a serine protease and several chitinases that involve in drastic degradation of the eggshell structure [262, 263]. A very similar mode of egg penetration is seen in fungi that are distantly related. Phylogenetic analysis of a chitinase gene from P. lilacinus with those from mycoparasitic, entomopathogenic and nematophagous fungi illustrated such similarity that it has been hypothesized that probably the gene was acquired by gene transfer from bacteria [264].

All zoospore producing species develop resting spores that survive in soil when their host is absent. These fungi colonize the female nematode and prevent cyst formation. Life cycle of N. gynophila is completed within 5 days at 13°C in the cereal cyst nematode [265]. The zoospores need flooded soil for motility, and therefore nematode infection is limited to periods following rainfall. It is difficult or impossible to culture the obligate parasites in vitro, therefore their commercial prospect is ambiguous [266].

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  • auli
    Can paecelomicis lila and meterhigium mix together with neem cake?
    6 years ago

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