Endoparasitic Fungi 4221 Introduction

Most of these fungi are obligate parasites and poor saprotrophic competitors in soil, but usually have a broad nematode host range. These obligate parasites live their whole vegetative life cycle inside their infected hosts [11, 16]. Endoparasitic fungi infect vermiform plant-parasitic nematodes using their spores (conidia or zoospores). The spores can be ingested by the nematode which germinate in the intestines (mostly the esophagus or mastax), or adhere firmly on the nematode cuticle when the nematode passes the fungus. The spore contents are inserted into the nematode by means of a narrow penetration tube, apparently with some mechanical pressure [18, 85]. Then an internal mycelium produces, and finally penetrates the cadaver to sporulate on its surface [20]. Some endosparasitic fungi produce zoospores that swim toward the nematode, attach to the cuticle usually around the natural orifices, and then encyst. The encysted zoospores penetrate the host body via those natural openings and start their vegetative growth. Afterward the hyphae develop some sporangium containing zoospores [11]. Taxonomy

We know a little about the actual taxonomy and phylogeny of this group of fungi. They are found in Blastocladiomycota (zoosporic Catenaria anguillulae), Haptocillium (formerly Verticillium), Harposporium (teleomorph: Podocrella) or Drechmeria [16]. In recent higher order classifications, posteriorly uniflagellate fungi (or chytrids) have been placed into three phyla: Blastocladiomycota, Chytridiomycota and Neocallimastigomycota. In this classification Catenaria spp. were transferred to Blastocladiomycota instead of previously accommodation under Chytridiomycota [86-90, 289]. The teleomorph of Nematoctonus (basidiomycetous Hohenbuehelia) contains both nematode-capturing and endoparasitic fungi [91].

Drechmeria was segregated from Meria [92] and its similarity with the Clavicipitaceae has been proven [93]. Some species demonstrate a continuum between the genera Harposporium and Hirsutella, developing two kinds of spores with the related kinds of conidiogenesis [94, 95]. The most distinctive nematophagous verticillium-like genera are Haptocillium species with adhesive conidia that stick to free-living nematodes [20]. Most Hirsutella species are entomogenous, and most of them are synnematous. No comprehensive revision of the genus has so far been published. A common nematode parasite of this genus is H. rhossiliensis [20]. Recently two endoparasitic fungi (Acrostalagmus bactrosporus and A. obovatus) with adhesive spores were transferred to the genus Haptocillium [96]. Ecology

Reports of endoparsitic fungi from different countries indicate a nearly cosmopolitan distribution, but a few species are either tropical or temperate. They were mostly described in the United States and Canada [20]. They were also reported from Ireland [97], New Zealand [98], El Salvador [99], and from plants and soils in the maritime Antarctic [26, 100, 288]. Drechmeria coniospora, Haptocillium balanoides, Harposporium anguillulae, and Hirsutella rhossiliensis were also isolated infrequently in Central America [101].

Maximum densities of Harposporium anguillulae are usually found in March and June in Swedish agricultural soil, where going down to 30-40 cm, while Hirsutella rhossiliensis strongly declines after 20 cm [20]. Densities of H. rhossiliensis in a Swedish agricultural soil peaked during September-November [54]. Population densities of these endoparasites specifically declined after fallow periods [54].

Nematode endoparasites were usually found in deciduous and conifer litter, old dung, moss cushions, and decaying vegetation [97]. Addition of farmyard manure to agricultural soil increased the population of endoparasites [102]. Nematodes are attracted toward Drechmeria coniospora, Haptocillium balanoides, and other endoparasites colonies [23, 69, 103-105].

In contrast with Hirsutella rhossiliensis whose conidia are infectious only while attached to a conidiophore, the conidia of Haptocillium species are equally infectious after liberation, and could bind to a rather wide range of nematode species. Consequently when H. balanoides applied as a suspension of conidia and hyphal fragments had a much greater effect than H. rhossiliensis in controlling Ditylenchus dipsaci and promoting growth of clover, both under gnotobiotic conditions [106] and in pot cultures [107]. According to these authors, H. balanoides has low sapro-trophic ability and does not survive in the soil for prolonged periods without added nematodes. Haptocillium is known to parasitize several nematode species, and with records to date, each Haptocillium species appears to have a limited degree of nem-atode host specificity [96]. Several species of nematodes such as D. dipsaci, Globodera rostochiensis and Panagrellus redivivus were inoculated with the fungus. Conidia adhered to all species but some of them were removed while the nema-tode moved through a layer of wet sand. Colonized individuals produced different quantities of conidia that were approximately 16,000, 11,700, and 840 for the above nematode species in the order mentioned [108]. Haptocillium bactrosporum,

Fig. 4.2 Sporulation of Haptocillium obovatum occurring externally

Fig. 4.2 Sporulation of Haptocillium obovatum occurring externally

Pasteuria Penetrans Fungal Colony

on hyphae constructed on head end of a nematode, arrowheads show some proliferating conidiogenous cells [96]

H. obovatum and H. balanoides all abundantly produce conidia on a bacterivorous nematode, Plectusi sp. (Fig. 4.2) [96].

Haptocillium balanoides was also founded on dead needle of Pinus densiflora in Tsukuba, Japan [109]. The difference between phytophagous and bacteriophagous nematodes is of great ecological importance in relation to endoparasitic and other nematophagous fungi. The host relation hypothesis proposed for endoparasitic fungi [110] is incompatible with the many reports of relatively little host specificity. The free-living stages of the same nematodes can be parasitized by different array of taxa, mainly Haptocillium and Hirsutella [20].

Additional examples of extensively studied endoparasitic fungi are Drechmeria coniospora, Nematoctonus spp. and Haptocillium balanoides [11]. Comparing with nematode-trapping fungi, such endoparasitic fungi are more amendable to practical application [54].

Hirsutella rhossiliensis was able to decrease nematode invasion, and therefore nematode populations of Meloidogyne javanica, Heterodera avenae, H. glycines and Criconema xenoplax were decreased. It also successfully infected several other species of Heterodera, Ditylenchus destructor, Meloidogyne hapla, Pratylenchus penetrans, Anaplectus granulosus, and even larvae of Globodera rostochiensis [111].

In an in vitro experiment, H. rhossiliensis killed Ditylenchus dipsaci in 4 days, and juveniles of M. incognita in 2 days [112]. Hirsutella rhossiliensis is considered responsible for rapid fluctuations of C. xenoplax populations in peach orchards [3, 113, 114]. Without nematodes as a food source, the population of H. rhossiliensis in soil dies out [115, 116]. Hirsutella rhossiliensis (18 isolates), H. minnesotensis (8 isolates) and H. vermicola (3 isolates) were compared for their nematode parasitism. Most isolates of H. rhossiliensis and H. minnesotensis parasitized higher percentages of the cyst nematodes (Heterodera glycines and H. avenae) than the four non-cyst nematodes (Meloidogyne hapla, Bursaphelenchus xylophilus,

Heterorhabditis bacteriophora, and Steinernema carpocapsae). Hirsutella vermicola had weak or no ability for parasitizing the six assayed nematode species [117].

The conidia of Hirsutella species are infective only when they are attached to the phialide [118], and furthermore, conidial germination can be greatly affected by soil fungistasis [113, 119]. Consequently the species seems less suited for biological control than species of Haptocillium [107]. No growth was observed below pH 5 on an agar pH gradient [120]. Except for isolates originating from Hoplolaimidae that grew more slowly, other different isolates had uniform characters of nematode pathogenicity. The Hoplolaimidae originated isolates had larger conidia, and were less pathogenic toward nematodes than isolates from other nematode hosts [121]. The fungus produced 78-124 conidia from a colonized individual J2 larva of Meloidogyne hapla, and caused a 50% decrease in J2 penetration of lettuce roots [122]. Patel et al. [123] succeeded to produce inoculum of H. rhossiliensis in liquid culture stirred in 5-L containers. Hirsutella minnesotensis is the second nemato-phagous species parasitizing the J2 of the soybean cyst nematode, Heterodera glycines [124]. The entomopathogenic species of Hirsutella did not attach to nematodes with their conidia and therefore had no controlling effect [112].

Hirsutella rhossiliensis is frequently seen in association with nematode populations and there are several reports on its suppression effect on populations of H. schachtti [125, 126] and potato cyst nematodes [127]. One worthy species for further investigation is H. rhossiliensis [11] with an obligate parasite lifestyle, make its population density related to population of its host nematode [115]. Contrasting with encouraging results of controlling nematodes by H. rhossiliensis in greenhouse and laboratory assays [128-131], the fungus did not decrease the population of cyst and root-knot nematodes in a number of field trials [132]. Because of the fungus inconsistent results, a better understanding of its ecology and population dynamics after being introduced into soil is critical for fungus successful use as an inundative commercial biocontrol agent.

A real-time PCR assay was developed to quantify the H. rhossiliensis [133] and H. minnesotensis [134] in soil. The results showed that the quantity of H. rhossiliensis DNA (according to real-time PCR) decreased over time (rapidly in the first 17 days and gradually for the succeeding 42 days), regardless of eggs or J2 of H. glycines as inoculum [135].

It is demonstrated that H. rhossiliensis could not decrease the M. javanica population on tomato over the long time [121], however, a related fungus, H. minnesotensis, was considered to have the ability of decreasing M. hapla population between 61% and 98% [136, 137].

Drechmeria coniospora can kill its host within 24 h and produce 5,000-10,000 conidia on each infected nematode [20]. Application of 106 conidia per 250-cm3 pot or 1,000 living infected Panagrellus redivivus as vectors could regulate Meloidogyne incognita in sterile or unsterile soil [138]. About 70% of nematodes which were inoculated with the conidia of D. coniospora retained attached condia after 16 h, with young ones being preferentially infected [139]. Positive correlation between reduction in spore adhesion and the nematode age increment has already been reported for Pasteuria penetrans [140].

Fig. 4.3 Adhesive conidia of Drechmeria coniospora on the head of a nematode. Arrow Adhesive bud of conidia, bar 2 ^m [143]

Fig. 4.3 Adhesive conidia of Drechmeria coniospora on the head of a nematode. Arrow Adhesive bud of conidia, bar 2 ^m [143]

Adding organic amendment to soil can indirectly increase the population densities of the fungus by stimulating bacteriophagous nematodes [141]. Application of D. coniospora as a biological control agent is not considered feasible, because its population density had not increased adjacent to plant roots; meanwhile the fungus has narrow host range that usually does not include the plant-parasitic nematodes [139, 141-143]. Mode of Action

Because of wider mouth openings of bacteriophagous nematodes which facilitate conidial ingestion, this group of nematodes are much more prone to parasitism by endoparasitic and nematode-trapping fungi. Chemical factors are responsible for the attachment of Drechmeria conidia (or to a lesser extent of Haptocillium conidia) to specific parts of the body [20]. The processes of D. coniospora conidiogenesis and penetration into nematode cuticle were illustrated by light- and electron-microscopy (Fig. 4.3) [143, 145].

Drechmeria coniospora secretes collagenase before and during penetration [146]. The fungus occupies the pseudocoelum of the nematode without colonization of the internal organs. Nematode can ingest the conidia, but no germination is seen in intestine [147]. Thus direct penetration of conidia through cuticle is the only way of infection.

Drechmeria coniospora attracts susceptible nematodes [103, 104]. The fungus develops teardrop-shaped conidia coated with a sticky mucous-like layer containing radiating fibrils [148, 149]. Conidia of the fungus stick to the nematode chemosensory organs, specifically in the mouth region and in male anal region of certain species. Infected nematodes lost their ability to respond chemotactically to all attraction sources [150] and they were no longer attracted by colonies of the fungus. The site-specific adhesion of conidia was demonstrated for bacteriophagous, a few plant-parasitic (Meloidogyne and Aphelenchus), and animal-parasitic nema-todes while there were no specific binding for some plant-parasitic nematodes like Pratylenchus, Ditylenchus and Criconemella species [143]. Conidia of D. coniospora can adhere to the chemosensory organs of root-knot nematodes but do not penetrate and colonize the nematode. Application of the fungus resulted in decreasing root galling in tomato roots, emphasized on involvement of chemo-tactic interference [138]. There are similar reports for insect-parasitic species (Neoaplectana and Heterorhabditis) [151] and Acrobeloides [139] where conidial adhesion occurs without any penetration.

It seemed that sialic acid-like carbohydrate (acetyl-neuraminic acid) which localized in head and tail regions, involve in binding to a lectin that located on the parasite's conidia. Treatment of spores with sialic acid and treatment of nematode with lectin Limulin reduced adhesion [150, 152]. Pronase treatment of the Caenorhabditis elegans also prevents adhesion of the conidia, but the nematodes regenerate the lost protein material after 2 h in Tris buffer [147]. The adhesion is also suggested to be mediated by sensilla exudates [147].

Adhesive on the conidial surface of D. coniospora always keeps its fibrillar appearance [78]. The fibrillar layer is dissolved in Pronase E. Infection was inhibited by Chymostatin (a protease inhibitor), suggesting the involvement of chymotrypsin-like proteases in the infection process [71]. After the binding of conidia of D. coniospora to nematode cuticle, an infection vesicle is developed within the cuticle layers [153, 154].

It is likely that a motile nematode previously colonized by D. coniospora, can be trapped by a second nematophagous fungus as well, however, penetration of Arthrobotrys oligospora to a D. coniospora colonized nematode is inhibited and its hyphae are often killed when placed adjacent to those of D. coniospora [85].

The uniflagellate zoospores of C. anguillulae attract toward natural openings (mouth, anus, excretory pores, etc.) of nematodes and after contacting with cuticle, show an amoeboid movement before encystment happen. A cell wall coated with a sticky material cover the encysting zoospores, and the flagellum is withdrawn. A penetration peg is developed from the encysted zoospore which breaches the nematode cuticle and usually invades and digests the nematode cuticle within 24 h. Then the hyphae develop some sporangium containing zoospores that can infect new nematode hosts after releasing. Ability to parasitizing nematode eggs is also reported for C. anguillulae [83].

Hirsutella rhossiliensis is a typical endoparasitic fungus of nematodes. It produces adhesive spores that attach to and penetrate the cuticle of passing nematodes [119]. The conidia are infectious if only they are attached to the phialides [118], and one conidium is generally enough to infect a nematode. When the fungus penetrates its host, the nematode will be totally colonized, and within a few days the new infectious conidia will be produced [155].

One neutral serine protease [156] and more recently a new extracellular alkaline protease (Hasp) [36] has been described from H. rhossiliensis. This enzyme was purified, cloned and examined against nematodes. Hasp could kill the juveniles of the soybean-cyst nematode (Heterodera glycines) after purification [36].

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