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Steinernema glaseri is known to be the most efficacious biocontrol agent against many soil insects belonging to various orders and of diverse habitats. In nature the efficacy of such entomogenous nematodes may be challenged by many micro pathogens. Keeping this in view, the biocompatibility of Steinernema glaseri with a nematophagous fungus Arthrobotrys superba was assessed on eight different nutrient media. The predatory activity of A. superba was evaluated in terms of trap formation, conidiophore formation, and number of adhesive cells formed in the presence and absence of nematodes. The fungus failed to form any trap on any of the culture media in the absence of nematodes. However, in the presence of nematodes, the trap formation by the A. superba was increased and found to be maximum on LNM (low nutrient mineral salt) medium and minimum at WA (water agar) of all the culture media tested. In the present investigation, the effect of CMA (2% and 5%) was not considerable; however, gradually higher dilutions of CMA induced greater number of traps. This less pronounced effect might be due to nutrient richness because low nutrient levels were found to be necessary for the formation of conidial traps. The number of conidiophores decreased with increase in dilution of Corn Meal Agar from 5% to 2%. Higher number of chlamydospores was observed in phenylalanine treated medium which indicates the inhibiting effect of phenylalanine on the growth of A. superba. Our results suggest that richness of soil has pronounced effect on the predatory activity of nematophagous fungus, Arthrobotrys superba which may further affect the biopotential of entomogenous nematode Steinernema glaseri, therefore, care should be taken while releasing entomogenous nematodes in an agroecosystem for managing various insect pests in a more efficient manner.

Pochonia chlamydosporia is a nematophagous fungus with high potential for the biological control of root-knot nematodes (Meloidogyne spp.). The fungus is commonly produced in colonised rice substrate, but non-staple and more affordable substrates may encourage the production and use of this biological control agent. An in vitro study was conducted to screen different organic materials, including green and animal manures and industrial by-products as substrates for local production of viable fungal chlamydospores. Chlamydospore yield from sugarcane filter mud, bean straw, sunn hemp, goat manure, African marigold, wild sunflower, maize cobs, and velvet beans substrates was equal to or significantly greater than production using rice (standard substrate). In addition, the percentage viability of chlamydospores produced in most organic substrates was greater than 50%. The use of non-staple substrates deserves further investigation as a viable, and economical local option to rice substrate for mass production of P. chlamydosporia inoculum in Sub-Saharan Africa.

Fusarium wilt is considered the main fungal disease of yellow passion fruit plants in Brazil. There is ample anecdotal evidence of greater intensity of Fusarium wilt after water shortage in field conditions, but this association needs scientific confirmation. There is also a need to increase the efficiency of inoculation with Fop (Fusarium oxysporum f. sp. passiflorae) under controlled conditions for research purposes. Therefore, in this study we evaluated the effect of propagation method (from cuttings or seedlings) associated with controlled water deficit on the incidence of Fusarium wilt in yellow passion fruit plants. The artificial inoculation with Fop involved application of a spore suspension of 106 conidia mL−1 and infestation of the potting media with Fop grown in sand and cornmeal substrate. For anatomical analysis, root segments were used from inoculated and non-inoculated plants (control). Seedlings that were submitted to water deficit presented the highest incidence of Fusarium wilt, 75.0%, while in the irrigated control the incidence was below 40.0%. The mortality associated with Fop in cutting-propagated plants did not differ from non-inoculated plants. The plants subjected to water stress had greater presence of hyphae and chlamydospores and reduced starch concentration in the root cortex region. Propagation by seeds associated with controlled water stress can be used to screen accessions of P. edulis for resistance to Fusarium wilt.

Duddingtonia flagrans is a natural strain of Nematophagous-Fungi isolated around the world. It has demonstrated efficacy and ease of use in laboratory as well as in field conditions. The fungus contributes to the prophylactic control of the worms by reducing the number of L3 on pasture. The aims of this study were to test and analyze the predatory effect of D. flagrans under sunny and shaded conditions on the L3 in the faeces, and to verify the reduction of translation to pasture during summer and winter seasons. Faecal Mass Units (FMUs) were assigned to two treated groups (groups treated with D. flagrans chlamydospores, TG) and two untreated groups (without D. flagrans chlamydospores, UG), in summer and winter, under sunny and shaded conditions. FMUs and herbage samples were taken for parasitological workup. Predatory activity of D. flagrans was evident under both conditions for the summer experiment but was not manifest for the winter experiment. In summer, an interaction between sunny and shaded conditions and predatory activity of D. flagrans was found. Environmental conditions on predatory activity should be considered when designing strategies for the implementation of D. flagrans in grazing systems to smooth the infectivity curve of L3. Fil: Bilotto, Franco. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina Fil: Fusé, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina Fil: Sagüés, María Federica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina Fil: Iglesias, Lucía Emilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina Fil: Fernández, Alicia Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina Fil: Zegbi, Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina Fil: Guerrero, Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina Fil: Saumell, Carlos Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina

Summary Invasive fusariosis has a high mortality and is predominantly observed in patients with leukemia. We report the first case of a novel species of Fusarium, Fusarium riograndense sp. nov, isolated from a lesion in the nasal cavity lesion of a patient with acute lymphoblastic leukemia. The etiological agent was identified by Multilocus Sequencing Typing (MLST), including RPB2, TEF-1a, and ITS-LSU sequences, the gold standard technique to identify new species of Fusarium. MLST and phenotypic data strongly supported its inclusion in the F. solani species complex (FSSC). The new species produced a red pigment in the Sabouraud Dextrose Agar similar to other members of the complex. The macroconiodia developed from phialides on multibranched conidiophores which merge to form effuse sporodochia with a basal foot-cell instead of papilla in basal cell shape. The microconidia were ellipsoidal, 0–1-septated, produced from long monophialides. Chlamydospores were produced singly or in pairs. Amphotericin B (MIC 1 µg/mL) was the most active drug, followed by voriconazole (MIC 8 µg/mL). The patient was successfully treated with voriconazole. Our findings indicate another lineage within FSSC capable causing of invasive human infection.

The present study aimed to evaluate different inoculation methods of Fusarium oxysporum f. sp. niveum in watermelon and the reaction of accessions from this crop. Firstly, seven inoculation methods using conidia were tested on the susceptible cultivar Sugar Baby, including the standard dipping method. The methods initially tested were not efficient; therefore, we tested a new methodology using chlamydospores which are fungus survival structures. After the production of chlamydospores in vermiculite enriched with liquid culture media potato and sucrose (PS), the new method was tested on an experiment comparing inoculation by conidia and chlamydospores. For the conidia method, the plants were inoculated after their final leaf formed. For chlamydospores, inoculations were done in plants or sowed seeds. The use of chlamydospores in sowed seeds was effective for the inoculation of Fusarium oxysporum f. sp. niveum in watermelon and showed the highest severity scores in relation to the others methods. The inoculation method using chlamydospores also obtained the shortest means of root and shoot length in the cultivars Charleston Gray and Sugar Baby. Thus, using this methodology, 25 accessions from the watermelon germplasm were evaluated 21 days after inoculation with a grading scale. Eight accessions were classified as resistant, corresponding to 32% of accessions evaluated. Eight other accessions received the highest score of severity, proving the efficiency of the methology to evaluate the reaction to the disease.

Equine gastrointestinal nematodiosis contributes to the lower productivity of these animals. There are growing reports of the emergence of nematodes resistant to the drugs used for decades in anthelmintic treatments. An alternative to the emergence of resistance may be the use of nematophagous fungi as a complementary method of treatment. Therefore, the objective was to evaluate the effects of the use of the product Bioverm® as a carrier of Duddingtonia flagrans, on pasture contamination level and equine parasitic burden. Sixteen mares were used, divided into two groups, one control and one treated, in which the treated animals received a dose of 1 g of Bioverm®, containing 105 chlamydospores per gram of the commercial product for each 10 kg of body weight, per day, for six months. Efficacy was evaluated by count of eggs per gram of faeces (EPG), coproculture and larval count on pastures and its correlation with climate, as well as weight gain evaluation. During the study, a significant influence of the formulation on weight gain and EPG was observed. The recovery of larvae from coprocultures revealed the predominance of small over large strongyles. There was a significant difference (p

Summary Introduction Candida africana and C . dubliniensis are closely related species of C. albicans . Current phenotypic methods are not suitable to accurately distinguish all the species belonging to the C. albicans complex. Several molecular-based methods have recently been designed for discriminating among closely related Candida species. The aim of this study was to establish the prevalence of C. dubliniensis and C. africana in vulvovaginal samples with phenotypic and genotypic methods. Materials and Methods We re-examined 376 vulvovaginal C. albicans complex isolates. All the isolates were identified with morphological features and HWP1 gene polymorphisms. ITS and D1/D2 sequencing, carbohydrate assimilation, MALDI-TOF MS profiles and antifungal susceptibilities were evaluated for C . africana and C . dubliniensis isolates. Results Of the 376 isolates, three C. africana and three C. dubliniensis isolates (0.8% and 0.8% prevalence, respectively) were identified by molecular methods (HPW1, ITS and D1/D2) Phenotypically, C . africana differed from C . albicans and C. dubliniensis by formation of no/rare pseudohyphae, absence of chlamydospores and, the development of turquoise green colonies on CHROMagar. MALDI-TOF MS and API ID 32 C could not revealed C. africana isolates. C. africana and C. dubliniensis isolates showed very low MIC values for all the tested antifungals. Discussion This first report of C. africana from Turkey provides additional data for epidemiological, phenotypic features and antimicrobial susceptibility profiles. This study also highlights the importance of using genotypic methods in combination with phenotypic methods.

Phytophthora ramorum is a concern of the ornamental nursery business because it is implicated in the movement on ornamental plants such as rhododendron and viburnum. Since there is a soil phase that infects the roots, above-ground symptoms are not often manifested and easily detected. Government agencies require that a P. ramorum-positive nursery be placed under some type of quarantine until it can demonstrate that it has mitigated P. ramorum. Typical methods of mitigation are often expensive, harmful to the environment, and impractical. Biological control using Trichoderma asperellum isolate 04-22 (Ta 04-22) has been demonstrated to reduce P. ramorum soil populations to below detectable limits when incorporated into soil or potting mix. The purpose of this study was to determine whether Ta 04-22 would reduce Viburnum tinus root infection and reduce secondary sporulation from infected roots. When rooted V. tinus cuttings were planted into potting mix amended with three different final concentrations (1 × 105, 1 × 106, and 1 × 107 CFU/cm3 potting mix) of Ta 04-22 and drenched with either a P. ramorum sporangia or chlamydospore suspension, plants with any infected roots declined as the Ta 04-22 concentration increased. This was significant (P = 0.039) when P. ramorum chlamydospores were used as the inoculum propagule but not (P = 0.143) when sporangia were used. A second application of Ta 04-22 as a top-dressing after 4 weeks did not result in a decrease in plants with infected roots. When P. ramorum-infected V. tinus roots were treated with Ta 04-22 grown on wheat bran, Ta 04-22 formulated as a wettable powder, or RootShield® Plus WP, flow-through of P. ramorum secondary inoculum was significantly reduced compared to non-treated controls (P

Phytophthora nicotianae causes root and crown rot disease in open field paprika pepper crops of Extremadura (western Spain). Sensitivity of the vegetative structures (mycelia and chlamydospores) of P. nicotianae to Brassica carinata pellets (Biofence) was evaluated in vitro at different doses (3, 6, 12 and 24 mg of pellet per plate) and temperatures (15, 20, 25 and 30 °C). The effectiveness of the pellets varied depending on the dose. An inhibition effect that decreased with time, was observed in the mycelial growth and chlamydospores germination in plate. The highest dose of pellets tested (24 mg) was fungitoxic to mycelium regardless of temperature for all the isolates. Moreover, biofumigation was effective to suppres chlamydospores germination when the pellets were incorporated into the soil (1.5 and 3 g L−1 of soil) under different temperature regimes. In bioassays with pepper plants, both rates of B. carinata pellets (1.5 and 3 g L−1 of soil) reduced populations of P. nicotianae below the limits of detection of our bioassay (

Phytophthora nicotianae is the principal causal agent of root and crown rot disease of pepper plants in Extremadura (western Spain), a spring-summer crop in this region. Preplant soil treatment by anaerobic soil disinfestation (ASD) may effectively control plant pathogens in many crop production systems, but field conditions and availability of C sources can limit its practical application. A laboratory experiment was conducted to study P. nicotianae control by ASD with low temperatures and several carbon (C) sources: rice bran, rapeseed cake, grape pomace and brewer’s spent grain. Survival and infectivity of pepper by P. nicotianae chlamydospores were reduced with all C sources assayed and redox potential in all ASD treatments indicated that reductive soil conditions were achieved. Rice bran (20 tons ha−1); rapeseed cake (20 tons ha−1), and grape pomace (40 tons ha−1) were also assayed in a field experiment in early spring. Survival and infectivity of P. nicotianae were also reduced with all C sources. An increase of dehydrogenase and urease activities and a strong pH decline were observed with rice bran and rapeseed cake, probably related to an increase of anaerobic bacterial populations in soil. Application of ASD with high C source rates may be effective in control of P. nicotianae under low temperature conditions.

The sclerotia of Villosiclava virens were found commonly in high altitude and the temperate regions, where the temperatures are relatively low in rice filling stage. To make sure if low temperature induce the sclerotial formation in V. virens, the inoculated rice panicles in laboratory and the diseased rice panicles cut from paddy fields were treated under different temperatures. The results showed that 3 days of night temperature at 15°C were enough to induce the sclerotial formation. The low temperature was much more effective for young balls with intact membranes. After appearance of chlamydospores on the ball surfaces, the sclerotium could not differentiate anymore. The sclerotia began to differentiate below the chlamydo-spore layer and gradually grew onto the ball surfaces. This suggests that low temperature in the early development stage of false smut balls is an important factor to induce the sclerotial differentiation, and rice cultivars with long growth periods are able to produce more sclerotium-bearing balls, which will produce mass of spores in paddy field in the coming year.

Co-cultivation is one of the significant ways to improve the efficacy of biocontrol agents. In the present study, the role of Vel1 on the sporulation, secondary metabolism, mycoparasitism and disease control of Trichoderma atroviride and its influence on B. amyloliquefaciens macrolactin and difficidin gene expressions during co-cultivation have been investigated through the knockout and overexpression of Vel1 gene. Under nutrient stress conditions the T. atroviride Vel1 knockout (T23ΔVel1) exhibited early chlamydospore differentiation, while wild type and over expression strains formed more chlamydospores under nutrient-rich conditions. The differential expression of genes related to sporulation, secondary metabolism, mycoparasitism, and disease control was upregulated by the Vel1 over-expression (T23OVel1), and co-cultivations (T23 + BA and T23OVel1 + BA). The expression of these genes was impaired by the deletion of Vel1 gene and co-culture of B. amyloliquefaciens and T23ΔVel1 strains. Interestingly, macrolactin and difficidin genes of B. amyloliquefaciens were downregulated during co-cultivation with T23OVel1 and upregulated during co-cultivation with T23ΔVel1 strains. The mycoparasitism, plant growth and bio-control activity of T23 + BA and T23OVel1 + BA were superior than other treatments. The T23ΔVel1 showed reduced activity of mycoparasitism, plant growth and biocontrol, but it was increased by the co-culture (T23ΔVel1 + BA) due to the enhanced activity of B. amyloliquefaciens. The results infer that Vel1 of T. atroviride is involved in sporulation, mycoparasitism, disease control and secondary metabolite production. Further, it confirmed that Vel1 downregulates macrolactin and difficidin gene expression of B. amyloliquefaciens and thereby it regulates the compatibility between the T. atroviride and B. amyloliquefaciens to control the wheat root rot disease caused by Fusarium graminearum.

This study evaluated the germination capacity of Pochonia chlamydosporia (VC4) fungus after its passage through the gastrointestinal tract of domestic chickens and its interaction with Ascaridia galli and Heterakis gallinarum eggs. Twenty-two domestic chickens were divided in two groups: control group (G1) received shredded corn substrate without VC4; and treatment group (G2) received a single dose of 29 g corn substrate containing 3.3 × 106 conidia/chlamydospores (VC4). Subsequently, chicken fecal samples were collected at intervals of 0, 6, 8, 10, 12, 18 and 24 h. Petri dishes from fecal samples of the treated group (G2) were subdivided (G2a and G2b), and then replicated in 2% agar-water medium for the microbiological test. After VC4 growth, approximately 200 eggs of A. galli (G2a) and H. gallinarum (G2b) were added to each subgroup to evaluation of ovicidal activity. There was fungal viability after passage through chicken gastrointestinal tract and egg predation of 59.9% and 43.2% for A. galli and H. gallinarum, respectively. The present work demonstrates the ability of the fungus P. chlamydosporia to survive after passing through the gastrointestinal tract of domestic chickens, an extreme environment (low pH, enzymes, microbiota and mechanical action), and still germinate after being excreted with feces.

Maize (Zea mays L.) is one of the important cereal crops in the world and is the third largest grown cereal crop in India. Field surveys conducted in 2013-15 recorded stalk rot incidence of 28–35% in southern states of India. The typical symptoms were observed after pollination with the drying of the lower leaves and eventually entire plant wilted prematurely, lower internodes turned in to grey-green color and stalks are hollow and weak leading to the lodging of the plant. Stalk rot associated pathogen was isolated on PDA medium. Out of 219 Fusarium isolates, 19 were distinct and the fungal colonies on PDA medium showed the development of pale brown to dark brown pigment. Macro conidia were produced in orange sporodochia from monophialides on branched conidiophores with apical cells tapered and elongated. Chlamydospores were solitary and intercalary. All 19 isolates were morphologically identical, and a representative isolate was used for molecular identification. The ITS rDNA and TEF gene were amplified and sequenced using ITS1/ITS4, TEF1/TEF2primer pairs. The nBLAST search and phylogenetic analysis confirmed that the pathogen was Fusarium equiseti. Pathogenicity tests conducted on 50-day-old maize plants by injecting conidial suspension of F. equiseti produced typical stalk rot symptoms after 15 days of post-inoculation and the pathogen's identity was confirmed by cultural and morphological features after re-isolation. Association of F. equiseti as the causal agent of sheath rot of maize was reported from China. The association of F. equiseti with stalk rot of maize is the first report in India.

Sterile carnation leaves are required for proper morphological identification of Fusarium spp. but the gamma irradiation equipment required for leaf sterilization is not available to everyone. This study evaluated three different methods for sterilizing carnation leaves: microwave radiation, ultraviolet, and chlorine gas (CG) sterilization. Both microwave and ultraviolet treatments did not sufficiently sterilize leaf tissue, however, exposure to CG for 2 h resulted in no growth of either fungi or bacteria. Exposure times of carnation leaves to CG were also evaluated for spore production, spore size and morphological characteristics of five Fusarium spp. Only carnation leaves exposed to CG for 45, 60 or 90 min were completely free of microorganism contamination. There were some differences in spore production and size, however, no differences were observed for characteristics essential for proper species identification such as micro- and macrospore features and production of sporodochia, perithecia, chlamydospores, and phialides for any of the CG exposure times. This study identified leaves sterilized by CG as a reliable substitute for gamma irradiation sterilization. The method described here is suitable for most laboratories and will provide a means for Fusarium identification when gamma irradiated leaves are not available.

Pochonia chlamydosporia is one of the most studied biological control agents of plant-parasitic nematodes. Soil amendment with agro-industrial residues may also suppress nematodes and improve plant growth. Therefore, the combined application of P. chlamydosporia and organic residues may enhance the control of nematodes. This study was designed to evaluate whether the integrated use of Pochonia chlamydosporia and the solid by-product of the wine industry (SBWI) controls Meloidogyne javanica. In greenhouse trials, SBWI (30 g kg−1 of air-dry soil) was applied to the soil with chlamydospores (500; 1500; 2500; 3500; 4500 and 5000 chlamydospores g-1 of air-dry soil) or colonised rice grains with mycelia and conidia of P. chlamydosporia (2.5; 5; 10 and 15 g kg-1 of air-dry soil). In laboratory, we assessed the effect of SBWI on the development of P. chlamydosporia. SBWI reduced the mycelial growth and sporulation of P. chlamydosporia in vitro. However, the integrated use of SBWI with the fungus increased tomato growth and reduced the number of galls and eggs of M. javanica per gram of tomato root in the greenhouse conditions, mainly when the fungus was applied at a rate of 3500 chlamydospores g-1 of soil and 15 g of colonised rice kg-1 of soil. In conclusion, the integrated use of P. chlamydosporia and SBWI controls M. javanica.

The non-pathogenic Fusarium oxysporum FO12 strain isolated from cork (Quercus suber) was one of the most effective treatments against Verticillium dahliae from among more than 200 natural compounds evaluated in previous studies. Based on the results of previous work, the present study aims to determine the potential of FO12 as a biological control agent against Verticillium wilt of olive (VWO). To this end, several in vitro and in vivo experiments were carried out to evaluate the effectiveness of FO12 against V. dahliae. Dual cultures were conducted, showing a significant reduction in the mycelial growth of V. dahliae. However, inhibition zones were not observed in vitro. Four different culture fractions of FO12 were prepared to evaluate their effect on the reduction of viable propagules of V. dahliae in soil and on the disease progression in inoculated plants. Treatments with crude culture broth of FO12 resulted in a total reduction of the inoculum density of V. dahliae in naturally infested soils as well as in the progression of VWO under controlled conditions. Treatments with conidial suspension or chlamydospores were also effective in reducing the inoculum density in the soil and the disease severity in olive plants. This work provides useful information about the efficacy of several FO12-based culture fractions against VWO under controlled conditions, which could be the basis for the development of effective commercial formulations in the coming future.

Piriformospora indica is a plant growth- and disease resistance-promoting fungus in the Sebacinales family. In this study, the effect of this symbiotic fungus on the growth of tissue-cultured banana seedlings was investigated to explore its potential applications in the banana tissue culture industry. P. indica plugs (P-1) and a P. indica chlamydospore suspension (P-2) were co-cultured with banana seedlings in rooting medium to investigate their growth-promoting efficiency. Both the P-1 and P-2 treatments improved the plant biomass and chlorophyll contents of banana seedlings. The P-2 treatment had stronger promoting effects than the P-1 treatment, especially on plant height and root length in the first 3 weeks. At 4 weeks after inoculation, seedlings in the P-1 and P-2 treatments showed increased plant height, more and longer roots, increased shoot fresh weight, leaf width and length, and stem circumference compared with controls. The P. indica treatments also significantly enhanced the chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid contents in banana leaves. The results of this study verified that the fungus can facilitate the growth of banana seedlings in vitro and shorten the rooting time of tissue-cultured banana seedlings. Therefore, it has the potential to improve the growth and quality of tissue-cultured banana plants.

Cotton is a globally important crop used for both its natural fiber and seed. Fusarium wilt, caused by the fungus Fusarium oxysporum f. sp. vasinfectum, is a major disease of cotton capable of causing significant economic loss. The fungus persists in soil as chlamydospores and in association with the roots of susceptible, resistant and non-cotton hosts as well as in seed. Management of Fusarium wilt is difficult and most successfully achieved through the use of resistant cultivars and pathogen-free cotton seed. Once inoculum has been introduced into the field, strategies such as soil solarization and fumigation are applied to manage inoculum levels. While experimentally successful, these techniques are of limited use in a commercial setting. Management of Fusarium wilt will continue to rely upon the development of new resistant cultivars. Additionally, understanding the mechanism of seed colonization and developing high throughput seed treatment and testing protocols will allow the industry to effectively avoid the introduction of inoculum.

Geohelminths are parasites that stand out for their prevalence and wide distribution, depending on the soil for their transmission. The aim of this work was to evaluate the predatory capacity of the fungal isolate of the genus Duddingtonia (CG768) on third stage larvae (L3) of Ancylostoma spp. in beach sand under laboratory conditions. In the assay A five treatment groups and 1 control group were formed. The treatment groups contained 5000, 10,000, 15,000, 20,000 or 25,000 chlamydospores of the fungal isolate and 1000 Ancylostoma spp. L3 in pots containing 30 g of sand. The control group (without fungus) contained only 1000 Ancylostoma spp. L3 and distilled water in pots with 30 g of sand. Evidence of predatory activity was observed at the end of 15 days, where we observed the following percentages of reduction of L3: Group 1 (4.5%); Group 2 (24.5%); Group 3 (59.2%); Group 4 (58.8%); Group 5 (63%). However, difference was noted (p

This study was done to evaluate the effect of the bionematicide Pc-10, based on Pochonia chlamydosporia var. chlamydosporia, on the control of Meloidogyne javanica. The fungus was applied by two methods: (1) drenching the substrate of seedlings with an aqueous suspension of Pc-10 (0, 4.5, 9.0, 13.5 and 18.0 g/L), or (2) incorporating Pc-10 into the soil of potted plants at a rate of 5000 chlamydospores/g of soil. The soil of each pot was infested with 3000 eggs of M. javanica, and one cucumber seedling was transplanted to each pot after 1 week. Increasing the doses of Pc-10 in the seedlings reduced the numbers of galls/g and eggs/g in the roots of cucumber plants in both experiments, regardless of the application method of the fungus in the soil. The application of 18 g/L of Pc-10 in the seedlings reduced the number of galls/g of roots by 46.04% and 49.44%, and the number of eggs/g of roots by 48.32% and 40.58% in the first and second experiments, respectively. Soil application of Pc-10 reduced the number of eggs/g of roots by 19.42% in the first experiment only. Drenching the substrate of seedlings with Pc-10 at 18 g/L controls M. javanica in cucumber and does not require additional fungus application in the soil.

The capability of helminth (nematode and trematode) parasites in stimulating nematode trap and chlamydospore development of the nematophagous fungus Arthrobotrys (formerly Duddingtonia) flagrans was explored. Dead adult specimens of trematodes (the liver fluke Fasciola hepatica and the rumen fluke Calicophoron daubneyi) and nematodes (the ascarid Parascaris equorum and the strongylid Oesophagostomum spp.), as well as their secretory products, were placed onto corn meal agar plates concurrently inoculated with A. flagrans. Trapping organs were observed after 5 d and chlamydospores after 16 d, including in the control plates in the absence of parasitic stimulus. However, our data shows that both nematodes and trematodes increase trap and chlamydospore production compared with controls. We show for the first time that significantly higher numbers of traps and chlamydospores were observed in the cultures coinoculated with adult trematodes. We conclude that both the traps and chlamydospores formation are not only related to nematode-specific stimuli. The addition of secretory products of the trematode C. daubneyi to culture medium has potential for use in the large scale production of chlamydospores.

Pochonia species can occur in many Meloidogyne- infested soils throughout the world. The objective of this work was to isolate Pochonia species from different Meloidogyne- infested soil locations in Brazil and to screen for potential isolates effective in controlling Meloidogyne javanica under glasshouse conditions. A total of 29 isolates were identified as Pochonia chlamydosporia . Among them, 65.52% were identified as P. chlamydosporia var. chlamydosporia and 34.48% as P. chlamydosporia var. catenulata. For the glasshouse experiments, fungal chlamydospores were incorporated into the soil (5000 g soil −1 ) and one tomato seedling was transplanted in 300 mL pot. After one week, each plant was inoculated with 1000 eggs of M. javanica. The isolates 1, 2, 3, 4, 9, 10, 21, 24, 28 (from Brazil) and 64 (from Spain) were the most efficient in reducing the number of eggs of the nematode. These isolates were re-evaluated in another glasshouse experiment, this time the number of eggs was increased (3000), inoculating both eggs and chlamydospores at the same time. After one week of inoculation, a tomato seedling was transplanted in each pot. In this experiment, isolates 64 and 10 were the most efficient in reducing the number of eggs by 72% and 60%, respectively.

The fallow period can represent an opportunity to farmers if the field can be trimmed to receive finishing cattle. Nematode control remains important at this stage, and the use of biological agents such as nematophagous fungi is desirable. Nonetheless, the indiscriminate use of fungicides on agricultural crops may affect the development and survival of fungal propagules. Therefore, we evaluated the in vitro activity of 14 agricultural fungicides against the Duddingtonia flagrans strain ARSEF 5701. The assays were based on CLSI M38-A2 broth microdilution and the agar diffusion method. The fungicides azoxystrobin, cyproconazole, cyprodinil and tebuconazole showed minimum inhibitory concentrations for D. flagrans below their respective maximum residue limits. Although complete radial growth inhibition was only observed for carbendazim at the maximum concentration tested, D. flagrans proved to be resistant to only two fungicides, cyprodinil and iprodione. The present study indicates that D. flagrans is sensitive to most of the common agricultural fungicides, and resistance depends upon the structure, i.e., hypha or chlamydospore. On the basis of these in vitro results, we concluded that the use of agricultural fungicides could seriously hamper the survival of the fungus D. flagrans in amended soils.

Batch cultivation of the fungus Piriformospora indica on Kaefer medium containing soluble starch resulted in a 1.5-fold improvement in amylase activity and a reduction in the time required for achieving maximum growth, amylase activity and spore yield by more than 50%. Peak growth (7.64 g/L) and amylase activity (39.84 U/mL) were recorded after 42 h while maximum spore yield (8.78×10 7 spores/mL) was achieved after 60 h of cultivation. The optimum temperature and pH for amylase activity was 80 °C and 7.0, respectively, with about, 80% of activity retained at 90 °C and 70% of activity retained at pH 9.0.

The control of gastrointestinal nematodes relies at present mostly on antihelmintic treatments using synthetic molecules. This approach, however, has led to the appearance of resistance to some types of antihelmintics which, together with the need to cut down on the use of chemicals, has fostered the development of other control methods, such as biological control, which is the use of living organisms that are naturally antagonistic to an unwanted species. Among the natural enemies of nematode parasitic larvae is the microfungus Duddingtonia flagrans. Research has shown the ability of this fungus to reduce the number of nematode larvae in faeces, the ability of its chlamydospores to survive the passage through the gastrointestinal tract of livestock and, moreover, to keep its germinative ability, thus facilitating the development of formulations. The present review looks at the species currently used and the different ways of administering already tested nematophagous fungi.

In the present paper, a novel method to accelerate sludge granulation is presented. Inoculation with chlamydospores of Phanerochaete sp. HSD accelerated sludge granulation during the treatment process of phenol wastewater, and the sludge granulation rate reached 66 ± 2% on day 7, 32 days earlier than that of the control inoculated with activated sludge only. Aerobic granule in R1 (AGR1) showed an annual ring-like multilayer structure and a primary core also existed in the nuclear area of the granule. The mechanism of rapid granulation revealed that the chlamydospore could survive in phenol wastewater and form the primary matrix on which aerobic granule was developed layer by layer. In addition, AGR1 developed in a phenol uptake system to counteract the adverse effects of phenol inhibition. Higher tolerance toward wastewater with high phenol strength was exhibited, and the maximum specific phenol degradation rate reached 1.54 g phenol g−1 VSS day−1.

The potential for using plant pathogens and seeds as indicator organisms for assessing sanitization of plant wastes during composting was tested in bench-scale flask and large-scale systems. Plasmodiophora brassicae was unsuitable due to high temperature tolerance in dry to moist composts, and detection of viable inoculum post-composting using bioassay plants not corresponding with that using TaqMan® PCR, possibly due to preservation of nucleic acids at elevated temperatures. Several other plant pathogens (Sclerotinia sclerotiorum, Microdochium nivale, Phytophthora cinnamomi and Phytophthora nicotianae) were unsuitable due their low temperature tolerance. Fusarium oxysporum f.sp. cepae and f.sp. radicis-lycopersici chlamydospores and tomato seeds were suitable indicators due to their moderate temperature tolerance and ease of viability testing post-composting. Abutilon seeds were more tolerant than tomato seeds of compost temperatures ≥52°C but more prone to degradation at lower temperatures and therefore less suitable as indicators. Relationships between compost temperature during exposures of 2-10 days and subsequent viability of the above chlamydospores or seeds enabled the sanitizing effect of composting processes to be predicted within 2-6 days. Plant waste type (woody or vegetable) had a small but significant effect on the relationship for tomato seeds but not for F. oxysporum chlamydospores.

Piptoporus quercinus, the rare oak polypore, is one of only four non-lichenised fungi listed on Schedule 8 of the Wildlife and Countryside Act 1981, in the UK. It is thereby accorded the highest level of legal protection in the UK. It fruits exclusively on oak (Quercus spp.), primarily veteran trees in old growth wood pasture and parkland in the midlands and southern England. Beyond information on fruit body occurrence, little is known of the ecology of this species. Germination of chlamydospores and basidiospores was investigated under a range of conditions (temperature, pH, different media). The mating system was ascertained by pairing single spore derived isolates, and somatic compatibility investigated by pairing fruit body isolates. Germination of basidiospores was consistently below 1 % with most success occurring on 2 % malt extract agar adjusted to pH 4 at 20 °C. Chlamydospores had a much higher average percentage of germination (47 % ± 22), and germinated even after prolonged exposure to extreme conditions (desiccation or exposure to −20 °C or 40 °C for 48 h, 7 d or 14 d). The mating system was unifactorial (bipolar), although only four mating alleles were discovered in the six populations sampled, two alleles being unique to one fruit body. There were no signs of somatic incompatibility between the majority of isolates, exceptions being an isolate from Wentwood, South Wales, and an isolate from Germany collected in 1975, both of which displayed apparent signs of somatic incompatibility in all experimental pairings. The lack of somatic incompatibility between the majority of UK isolates, together with the low number of mating alleles, may point to inbreeding within the UK population. Apparently compatible isolates were not, however, clonal, as shown by variation in their mycelial extension rates on agar at various temperatures.

The influence of ambient conditions on the development of Metarhizium anisopliae chlamydospores in tick eggs is reported for the first time. The infection of tick eggs by M. anisopliae involves common events, such as adhesion, conidial germination, appressoria formation, invasion, and development within the eggs. However, the final stage of fungal development differs according to the environmental conditions. At high humidity (close to 100%) and moderate temperature (25°C) the fungus emerged from the eggs and formed conidiophores and conidia externally on the dead eggs. Elevating the temperature to 30°C or reducing humidity to 55-75% induced the production of chlamydospores inside the eggs, without conidiogenesis. When eggs with mature chlamydospores were returned to the appropriate conditions (25°C and 100% RH), conidiogenesis was recovered. Formation of chlamydospores, observed by means of histology and TEM, began with the thickening and septation of hyphae. As the chlamydospore wall thickened a new external undulated wall layer appeared. The mature chlamydospore in eggs has an oval shape (5.3 ± 0.9 microm long, 2.5 ± 0.2 microm wide); its wall comprises three distinct layers. The ability of M. anisopliae to produce chlamydospores under harsh conditions is advantageous and should be considered in application.

The response of soil fungi to the insecticide Reldan (chlorpyrifos-methyl) was studied under field and laboratory conditions. The colony forming units (CFU) of fungi were determined by means of serial dilution technique after 1 day, 7, 14, and 28 days of the insecticide application in soil. The plate count data indicated that the insecticide affected the number of fungi in treated soil compared with the untreated soil; the developed colonies decreased after 1–7 days of insecticide application and then increased with increasing application time. The effect of different concentrations of insecticide on the morphological features and sporogenesis of Aspergillus flavus , Fusarium culumorum , and Drechslera biseptata was studied. Relative conidiospore formation was sharply decreased and the conidiophores were deformed in A. flavus , while F. culumorum and D. biseptata failed to produce conidiospores, although chlamydospores were formed in large numbers when cultivated on the growth medium containing insecticide.

The effect of different parameters, including the type of nutrients, the quality of the gelling agent, and the genotype of the strain, were evaluated in the production of gametangia by Phytophthora ramorum in vitro . By comparing different agar sources on a carrot-based medium, a delay or a failure in the production of oospores was observed in pairings carried out on media supplemented with technical agar. In contrast, oospores were produced on other agar types, the production on media supplemented with agarose being slightly higher. The formation of gametangia was also influenced by the genotype of the strains involved in the pairing. A European A1 strain producing very few chlamydospores was found to be a better mating partner than other A1 strains. Using a carrot–agarose medium and selected genotypes, all European isolates were characterized in terms of mating type. A macroscopic experiment highlighted a particular spatial distribution of P. ramorum oospores in vitro . A method using polycarbonate membrane was evaluated to assess the selfing ability of P. ramorum .

Difference in expression of putative virulence factors and in antifungal susceptibility among different Candida species has raised the need for species-level identification. The close relationship of Candida dubliniensis with C. albicans has led to misidentification of C. dubliniensis isolates as C. albicans . Phenotypic tests include ability to produce chlamydospore on casein agar, colony colour development on differential media CHROM agar Candida medium and ability to form hyphal fringe on Pal's agar, have been used to differentiate these two Candida species. Fifty isolates of Candida species were recovered from various specimens (blood, urine, tissue and respiratory secretions) from diabetic and cancer patients between April and July 2007. The isolates were tested for chlamydospore production on casein agar. These were also streaked simultaneously on CHROM agar, Pal's agar and a combination of CHROM agar supplemented with Pal's agar for identification and differentiation of C. dubliniensis from C. albicans . On CHROM agar, 19 isolates were identified as C. dubliniensis , nine as C. albicans , 10 as C. krusei , nine as C. tropicalis and two as C. glabrata . One was indeterminate and later identified as C. dubliniensis . Out of the 20 C. dubliniensis isolates, 19 isolates exhibited hyphal fringe on Pal's agar. On CHROM agar supplemented with Pal's agar, 16 out of the 19 fringe-positive isolates exhibited fringe surrounding the bluish green-coloured colonies of C. dubliniensis . Additional identification tests like growth at 45oC and ability to reduce 2,3,5-triphenyltetrazolium chloride were time efficient, inexpensive and easy-to-use methods for differentiation of C. dubliniensis and C. albicans isolates. CHROM agar when supplemented with Pal's agar gave definitive identification between C. dubliniensis and C. albicans .

Previous observations showed that Duddingtonia flagrans chlamydospores were visualized in McMaster chambers containing faeces of treated sheep. This trial explored the McMaster technique as a tool to quantify chlamydospores in sheep faeces. A range of individual chlamydospore doses (from 19.5 x 10(6) to 177.5 x 10(6)) were offered orally to nine lambs for 7 consecutive days. A faecal sample (5 g) was daily obtained from the rectum of each animal (from days 1 to 13) to perform the McMaster technique using a sugar flotation fluid with 1.27 g/mL density. Each chlamydospore counted in the McMaster chamber was considered as 50 chlamydospores per g of faeces (CPG). The results confirmed that the estimated CPG was associated with the daily dose offered to the animals (r(2)=0.90; P0.001). Furthermore, the total chlamydospore dose received by each animal was strongly associated to the total quantity of CPG obtained from the bulk faeces (TCtot) (r(2)=0.96; P0.0001). Quantification of CPG can be used as a helpful tool to determine the number of chlamydospores reaching the faeces in orally dosed animals. This could be used to evaluate the efficacy of D. flagrans for the control of gastrointestinal nematode larvae in sheep faeces.

A less resource intensive procedure was used to select an endophytic non-pathogenic isolate of Fusarium oxysporum (NP Fo ) and an antibiotic producing rhizosphere/rhizoplane (RS-RP) competent fluorescent pseudomonad to suppress Fusarium yellow ( F. oxysporum (Schlecht) f. sp. phaseoli Kendrick and Snyder) of bean ( Phaseolus vulgaris L.). Using the combined criteria of in vitro and in planta competitiveness resulted in selection of three isolates for determination of their disease suppressing attributes. Several isolates of fluorescent pseudomonads obtained from RS-RP and internal root tissues of bean were tested for antibiosis in vitro. The isolates producing large inhibition zones were selected to determine RS-RP competence through seed treatment. The three most competent isolates were tested for their capacity to reduce root colonization by the pathogen, which resulted in the selection of only one isolate, identified as Pseudomonas putida (Trevisan) Migula biotype A. The NP Fo isolates Fo10 and Fo6 endophytically occupied about 80% of the root length compared to only 60% by the isolate Fo16. Mixing chlamydospores of Fo10 in non-sterile field soil, reduced RS-RP and internal root colonization by the pathogen by 95 and 89%, respectively. In the greenhouse trial, mixing chlamydospores of either of the three NP Fo isolates, the area under the chlorophyll retention curve (AUCRC) increased significantly with Fo10 compared to Fo6 or F16, over the positive control. The grain yield/plants from Fo10 treated soil was 177 or 291% higher than from Fo6-soil or positive control, respectively, but remained 50% lower than in pathogen-free negative control soil. In the field plots with pathogen ID of 850 ± 150/g soil, mixing chlamydospores of Fo10 (4250 ± 113/g), increased AUCRC and grain yield by about 100% over the control. However, if the bean seeds were treated with P. putida , the grain yield increased by 211% without further increase in AUCRC. The suppressive effect of Fo10 on disease development appeared to be related to its high saprophytic competitiveness with the pathogen in the soil matrix, which resulted in lower ID in the root zone, and parasitic competitiveness by reducing the availability of infection sites and internal root colonization by the pathogen. Seed treatment with P. putida provided additional protection in the root zone.

Over the last 35 y, the life cycle of endosymbiotic gut fungi (Harpellales) has been expanded to include cyst stages associated with the developing ovaries. Ovarian cysts (chlamydospore stages) have been identified after germination and production of asexual trichospores in vitro, but germination is not always successful, and spores exhibit morphological variation. Sequence data (for partial 18S and 28S rRNA genes) were generated for these putative stages of Harpellales using ungerminated cysts from adult blackflies and germinated cysts associated with field-collected blackfly egg masses. Cladistic analyses of the 18S and 28S rRNA sequences confirmed that ovarian cysts are stages in the life cycle of Harpellales. Ungerminated cysts, from a blackfly collected from New York state were identified as Pennella simulii and two samples from Newfoundland Prosimulium mixtum adults were identified as Harpella melusinae. Cysts with bipolar germ tubes, associated with field-collected Simulium egg masses from Newfoundland, were also identified as H. melusinae. Two other samples of cysts could not be matched with available sequences of gut fungi from larval hosts. The potential use of this approach to identify pathogenic stages associated with adult ovaries or field-collected egg masses among other host groups is highlighted and promoted as a tool to test the hypothesis that ovarian cysts are a dispersal stage common to all genera of Harpellales.

The oomycete ( Peronosporomycete ) Chlamydomyzium oviparasiticum , previously recorded as a parasite of rotifer eggs, was found infecting Rhabditis nematodes in a sample of rotting garden compost. For the first time C. oviparasiticum was cultured in liquid media, which enabled more detailed studies of zoospore behaviour and facilitated the use of confocal microscopy. Rhabditis nematodes were successfully re-infected from liquid-cultured inoculum. Light (including video) microscopy and transmission electron microscopy were used to document details of thallus development, zoospore release and resting spore morphology to enable comparison with other oomycete species. This species showed several significant saprolegnialian characters such as the ‘achlyoid’ pattern of spore formation, centrifugal cleavage and structured encystment vesicles. In contrast, spore release into a transient vesicle was a peronosporalean characteristic. The thick-walled resting spores showed relatively poor cytoplasmic preservation and had a thick multi-layered wall. It was still not possible to unequivocably decide whether these were chlamydospores or parthenogenically formed oospores. The phylogenetic significance of these observations is discussed.

Candida species are responsible for 80% of all nosocomial fungal infections. In 1995 a new yeast species was described, Candida dubliniensis which shares with Candida albicans characteristics. We have studied 109 yeast isolates identified as C. albicans to investigate the presence of C. dubliniensis by microbiological studies and PCR using DUBR/DUBF primers. Positive results using microbiological tools were between 90 and 98%. Two morphological and physiological of the 80 DNA examined samples (2.5%) showed a PCR product of 288 bp which allow the identification of C. dubliniensis. This is the first report in Venezuela of identification of this species using a PCR approach.

Diverse effects of two temperature regimes (20 and 30 °C) on the growing rates of five Duddingtonia flagrans isolates (MUCL 28429, CBS 143.83, CBS 561.92, CBS 565.50, and CBS 583.91) propagated on two liquid (MM, LB) and four solid substrates (CMA, SAB, SAB-GM, and SAB-HP) were observed. All D. flagrans isolates were able to produce chlamydospores but not on all substrates. None of the isolates produced trapping nets and conidia under applied growing conditions. D. flagrans isolates showed moderate insecticidal properties against Galleria mellonella larvae with mortality rates below 20%. Preincubation (18 h) of Heligmosomoides polygyrus infective (L 3 ) larvae in fungal homogenates highly impaired in vitro spontaneous motility of nematodes. This may indicate the potential of D. flagrans bioactive substance(s) for use as biocontrol agents of parasitic nematodes.

The lack of an adequate shelf-life in mycoherbicidal products has been an obstacle to their commercialization. Therefore, experiments were conducted to study the effect of inoculum type and concentration on the viability of the encapsulated propagules of Fusarium oxysporum , abbreviated as Foxy 2, in Pesta granules during storage. Pesta granules were made with different inocula of Foxy 2, including: microconidia, a mixture of mycelia and microconidia, and fresh as well as dried chlamydospore-rich biomass, each with three different inoculum concentrations. All granular preparations (0.5–2 mm, particle size) were stored at 4 °C or at room temperature (21 ± 3 °C) for 1year. All Pesta granules containing chlamydospore-inoculum retained higher viability (up to 100%) than those with mycelial and/or microconidial inocula, irrespective of inoculum concentration and storage temperature. Microconidial and mycelial preparations were not viable after 1 year at room temperature. Throughout the year, the viability of fresh and dried chlamydospore-rich biomass was not significantly affected by the concentration of chlamydospores in the formulation at 4 °C or room temperature. Thus, 85–100% viability of Foxy 2 propagules can be achieved in Pesta granules (0.5–2 mm) for at least 1 year by formulating chlamydospore-rich biomass and storing at a temperature of 4 °C. This information has significant implications in enhancing shelf-life of Foxy 2 products thereby helping us to overcome this obstacle to commercialization.

Chlamydospore formation from mycelia and conidia of Cylindrocarpon destructans isolated from the root rot lesion of the Panax ginseng was investigated by scanning electron and light microscopy. Typical chlamydospores were formed only from hyphae but not from conidia on culture media. However, immature chlamydopspore-like cells were formed from microconidia after 12 days of incubation at 20℃ on Czapek Dox broth (CDB) adjusted to pH 4.0. Chlamydospores were yellowish or reddish brown in color, and produced singly or in chain with the hyphal intercalary or terminal position on potato-dextrose agar, V-8 juice agar and CDB with no addition of nitrogen sources after 16~20 days of incubation at 20℃. They were 11.3 to 11.9 ㎛ in diameter, having many lumps-like warts on their surface with the length of 1.5 to 1.8 ㎛.

The results reported in this paper represent work from two separate experiments, namely a plot trial using cattle feces conducted at Kungsangen in Uppsala, Sweden and a plot trial using sheep feces undertaken at Tastrup in Copenhagen, Denmark. In both trials, a technique was used to monitor the level of Duddingtonia flagrans propagules in soil surrounding feces. The feces were from animals fed or not fed D. flagrans fungal chlamydospores. Also presented are the numbers of soil nematodes in soil surrounding sheep feces. The results indicate that D. flagrans has little growth beyond the fecal environment into surrounding soil when chlamydospores are fed to either sheep or cattle. This is substantiated by the soil nematode data. No statistical differences in the number of nematode taxa identified, Shannon Weiner H′, proportion of various feeding groups, and B/B + F (B and F are the proportions of bacterial and fungal-feeding nematodes) were found when soil surrounding sheep feces containing chlamydospores and parasitic nematode eggs was compared to soil surrounding feces containing parasitic nematode eggs alone. It is unlikely that the application of D. flagrans as a biological control agent against the free-living stages of nematode parasites of these livestock will negatively affect populations of nontarget soil nematodes.

Three different inoculum forms of Trichoderma koningii were tested in vitro for their ability to parasitize the sclerotia of Sclerotium rolfsii . Tests were conducted under two temperature regimes and three incubation periods. Wheat bran was proved to be the most potent inoculum form of the antagonist in reducing 0the viability of the sclerotia. Microscopical observations revealed the presence of hyphae, chlamydospores and conidia of T. koningii in the medullar tissues of the sclerotia. This is the first report of the effect of different inoculum forms of T. koningii on the sclerotia of S. rolfsii and of propagule (chlamydospores and conidia) formation of the antagonist inside the sclerotia of S. rolfsii .

Merimbla humicoloides sp. nov. was isolated after heat treatment of soil collected in a pine forest of Veracruz state, Mexico. The fungus is characterized by pale buff to cinnamon colonies that become dark brown to black in reverse and conidiophores that vary from irregularly asymmetrical penicilli to symmetrical penicilli with inflated metulae, and Humicola-like chlamydospores on the submerged hyphae. Phylogenetic inferences made from the ITS1-5.8S-ITS2 rDNA sequences indicate that M. humicoloides is a member of the Trichocomaceae and that it is related to M. ingelheimensis, Hamigera avellanea, and Penicillium species.

Rhynchosporium alismatis , a pathogen of several Alismataceae species, is being studied in Australia as a potential weed biocontrol agent for Alisma lanceolatum, A. plantago-aquatica and Damasonium minus. Chlamydospores of R. alismatis are described for the first time. Large numbers (5.34 × 10 5 cm −2 ) were produced on potato dextrose agar within 8–15 d and range in size from 4.7 to 14 μm. There was variability between isolates in mean diameter but size was not related to the host of origin. Within 24 h at 30 °C 60% of chlamydospores, 8 d to 3 months old, germinated. Each germinated chlamydospore produced up to four germ-tubes. Chlamydospores were detected in leaf lesions on naturally-infected A. plantago-aquatica. One-month-old chlamydospores were pathogenic to leaf discs cut from mature leaves of A. lanceolatum and Damasonium minus. The relative robustness of chlamydospores is an advantage over conidia for the future production of a biological control agent if they can be produced and harvested in broth culture.

Mortierella tsukubaensis sp. nov., isolated from uncultivated soil at Tsukuba, Japan, is described and illustrated as a new homothallic species. Simple, large sporangiophores with terminal, many-spored sporangia, large chlamydospores and naked zygospores are characteristic of M. tsukubaensis. A key of the 13 known homothallic species is provided.

New patterns of conidial formation on basidiospores and vegetative hyphae are identified, illustrated and described. The mechanisms of conidial ontogeny so far unsatisfactorilly studied are elucidated and now better understood. Several types of conidiogenous cells are described and their ontogeny better illustrated and explained. Arthroconidia of the fungi are for the first time studied at ultrastructural level and their mode of formation and secession as well better understood. Likewise, chlamydospores of D. stillatus are for the first time investigated at ultrastructural level, what enables a better observation of their structure and morphology and as well a better understanding of their origin and various modes of formation.

Duddingtonia flagrans was grown in shake flask culture on Sabouraud's dextrose medium with and without the addition of agar (to form a slightly more viscous medium). The addition of agar increased the number of chlamydospores produced in late stationary phase, with 8.6 × 10 5 chlamydospores ml −1 being obtained on a medium containing 5 g agar l −1 . The pH optimum for growth was between 5.5–7.5 but colonies formed on plate cultures buffered at pH 10.5. When grown in shake flask culture at 25°C and pH 7 on malt extract-yeast extract-peptone-glucose medium (MYPG), D. flagrans had a specific growth rate of 0.21 h −1 (doubling time 3.3 h) and produced 5.1 ± 0.07 mg biomass ml −1 . To increase chlamydospore production in a liquid medium, D. flagrans was also grown in a two phase culture system in which late exponential phase biomass produced in MYPG medium was transferred to Vogel's mineral salts medium containing various nitrogen (mycological peptone, casein) and carbon (sodium acetate, starch and glycerol) sources. The highest chlamydospore concentrations (6.2 × 10 5 and 5.6 × 10 5 ml −1 ) were observed when the biomass was transferred to Vogel's medium containing 10 g starch or glycerol l −1 , respectively.

To determine the factors affecting the germination, early germ-tube growth (collectively called ‘germination’) of chlamydospores of Fusarium oxysporum f. sp. cubense and the severity of Fusarium wilt in banana plantlets, we varied chemical and physical factors in a suppressive and conducive soil. Soil temperature (4–40°C), water content (40–80% field capacity), and pH (4–10) were varied, and various amounts of CaCO 3 , Ca(OH) 2 and Fe-EDDHA were added to the soils. The suppressive soil had higher populations of actinomycetes and bacteria than the conducive soil that had higher populations of filamentous fungi and yeasts. The suppressive soil reduced germination of chlamydospores by 41±4%, and more than halved disease severity in banana plantlets ( Musa spp., AAA, Cavendish subgroup, cv. Williams). Soil water content, from wilting point to 80% field capacity, had little effect on germination of chlamydospores in either soil. In contrast, a water content of 40% field capacity promoted disease severity in both soils, compared with wetter soil. Increasing the temperature from 20 to 30°C in the conducive soil increased chlamydospore germination by 1.9 fold, but in the suppressive soil it had a much less effect (1.2 fold increase). This indicated that a physical process, such as diffusion, is likely to limit germination in the suppressive soil. An increase in temperature from 24 to 34°C increased disease severity in both suppressive and conducive soils. At 14°C no wilt symptoms were evident but the pathogen was recovered from within the plant. Chlamydospore germination was greatest in both soils at their natural pH of 8. A similar pattern existed for disease severity in the conducive soil. However, in the suppressive soil pH had little effect on disease severity. Adding CaCO 3 , Ca(OH) 2 , CaSO 4 or Fe-EDDHA to the soil reduced germination and disease severity by one-third to one-half in both soils. Smaller amounts had the greatest effect and the amounts of Ca compounds used were insufficient to change soil pH. The effects of manipulating soil pH, Ca and Fe content, temperature and water content on disease expression in the field need to be demonstrated.