Mycoparasitic Ability Research Articles

Serine protease CrKP43 interacts with MAPK and regulates fungal development and mycoparasitism in Clonostachys chloroleuca.

Serine proteases are a group of important hydrolytic enzymes that play vital roles in various cellular processes in fungi. In this study, the S8 serine protease-encoding gene CrKP43 was identified in the highly efficient Clonostachys chloroleuca 67-1 (formerly C. rosea 67-1) strain, which was markedly upregulated when parasitizing Sclerotinia sclerotiorum. The function of CrKP43 was investigated using gene deletion and complementation, and the results indicated that the lack of CrKP43 resulted in deformed fungal hyphae and cell morphology, inhibition of conidiation, and decreased antagonistic activity toward the pathogenic fungus Fusarium oxysporum f. sp. cucumerinum. Moreover, the mutants displayed much weaker mycoparasitic ability to S. sclerotiorum sclerotia and lower control efficiency against soybean Sclerotinia rot compared with the wild-type strain. All biological characteristics and biocontrol activities were recovered when the CrKP43 gene was reinserted into the fungus. Using qRT-PCR analysis and protein-protein interaction assays, it was further proved that the CrKP43 protein interacted with the mitogen-activated protein kinase (MAPK) Crmapk, suggesting serine proteases might be involved in the mycoparasitism of C. chloroleuca with the regulation of Crmapk. The findings improve our knowledge of serine proteases and their regulation in mycoparasites and help to illuminate the mechanisms underlying mycoparasitism of C. chloroleuca. IMPORTANCE Mycoparasites play important roles in the biocontrol of plant fungal diseases, during which they secret multiple hydrolases such as serine proteases to degrade their fungal hosts. In this study, we demonstrated that the serine protease CrKP43 was involved in C. chloroleuca development and mycoparasitism with the regulation of Crmapk. To the best of our knowledge, it is the first report on the functions and regulatory mechanisms of serine proteases in C. chloroleuca. Our findings will provide new insight into the regulatory mechanisms of serine proteases in mycoparasites and contribute to clarifying the mechanisms underlying mycoparasitism of C. chloroleuca, which will facilitate the development of highly efficient fungal biocontrol agents as well.

Glucose-6-phosphate 1-Epimerase CrGlu6 Contributes to Development and Biocontrol Efficiency in Clonostachys chloroleuca.

Clonostachys chloroleuca (formerly classified as C. rosea) is an important mycoparasite active against various plant fungal pathogens. Mitogen-activated protein kinase (MAPK) signaling pathways are vital in mycoparasitic interactions; they participate in responses to diverse stresses and mediate fungal development. In previous studies, the MAPK-encoding gene Crmapk has been proven to be involved in mycoparasitism and the biocontrol processes of C. chloroleuca, but its regulatory mechanisms remain unclear. Aldose 1-epimerases are key enzymes in filamentous fungi that generate energy for fungal growth and development. By protein-protein interaction assays, the glucose-6-phosphate 1-epimerase CrGlu6 was found to interact with Crmapk, and expression of the CrGlu6 gene was significantly upregulated when C. chloroleuca colonized Sclerotinia sclerotiorum sclerotia. Gene deletion and complementation analyses showed that CrGlu6 deficiency caused abnormal morphology of hyphae and cells, and greatly reduced conidiation. Moreover, deletion mutants presented much lower antifungal activities and mycoparasitic ability, and control efficiency against sclerotinia stem rot was markedly decreased. When the CrGlu6 gene was reinserted, all biological characteristics and biocontrol activities were recovered. These findings provide new insight into the mechanisms of glucose-6-phosphate 1-epimerase in mycoparasitism and help to further reveal the regulation of MAPK and its interacting proteins in the biocontrol of C. chloroleuca.

The fungus Clonostachys epichloë alters the influence of the Epichloë endophyte on seed germination and the biomass of Puccinellia distans grass.

The fungal grass endophyte Epichloë typhina (Pers.) Tul. & C. Tul. (Ascomycota: Clavicipitaceae) grows intercellulary in aerial plant parts and reproduces asexually by invading host seeds. In this phase, it enhances seed production and germination, which accelerates its vertical spread. This relationship may be distorted by other seed-born fungi, whose spread is not so directly dependent on the success of the grass. Recently, the fungus Clonostachys epichloë Schroers has been observed on Puccinellia distans (Jacq.) Parl seeds originating from grass clumps infested with stromata, sexual structures of Epichloë typhina that are formed in spring on some host culms, preventing flower and seed development ('choke disease'). C. epichloë shows mycoparasitic activity toward Epichloë stromata by reducing the production of ascospores, which are responsible for horizontal transmission of the fungus. The aim of this study was to investigate the effect of seed-borne C. epichloë on seed germination, as well as the size and weight of P. distans seedlings and to examine whether C. epichloë alters the influence of Epichloë in the early developmental stages of P. distans. The results showed that if C. epichloë acts on seeds together with E. typhina endophytes, the seeds were negatively affected due to the elimination of the positive effect of the latter in terms of both seed germination rate and seedling length. At the same time, C. epichloë increased the proportion of E. typhina-untreated germinated seeds. Additionally, only the joint action of the two fungi, E. typhina and C. epichloë, effectively stimulated seedling dry mass; the presence of E. typhina alone was not sufficient to noticeably affect seedling size. Based on the increasing commonality of C. epichloë on Epichloë stromata, as well as its potential to be used in biocontrol of 'choke disease', we should take a closer look at this fungus, not only in terms of its mycoparasitic ability, but also in terms of its cumulative impact on the whole Epichloë-grass system.

What is the role of the nitrate reductase (euknr) gene in fungi that live in nitrate-free environments? A targeted gene knock-out study in Ampelomyces mycoparasites

Mycoparasitic fungi can be utilized as biocontrol agents (BCAs) of many plant pathogens. Deciphering the molecular mechanisms of mycoparasitism may improve biocontrol efficiency. This work reports the first functional genetic studies in Ampelomyces, widespread mycoparasites and BCAs of powdery mildew fungi, and a molecular genetic toolbox for future works. The nitrate reductase (euknr) gene was targeted to reveal the biological function of nitrate assimilation in Ampelomyces. These mycoparasites live in an apparently nitrate-free environment, i.e. inside the hyphae of powdery mildew fungi that lack any nitrate uptake and assimilation system. Homologous recombination-based gene knock-out (KO) was applied to eliminate the euknr gene using Agrobacterium tumefaciens-mediated transformation. Efficient KO of euknr was confirmed by PCR, and visible phenotype caused by loss of euknr was detected on media with different nitrogen sources. Mycoparasitic ability was not affected by knocking out euknr as a tested transformant readily parasitized Blumeria graminis and Podosphaera xanthii colonies on barley and cucumber, respectively, and the rate of mycoparasitism did not differ from the wild type. These results indicate that euknr is not involved in mycoparasitism. Dissimilatory processes, involvement in nitric oxide metabolism, or other, yet undiscovered processes may explain why a functional euknr is maintained in Ampelomyces.

Characterization of a novel dsRNA mycovirus of Trichoderma atroviride NFCF377 reveals a member of "Fusagraviridae" with changes in antifungal activity of the host fungus.

Trichoderma atroviride is a common fungus found in various ecosystems that shows mycoparasitic ability on other fungi. A novel dsRNA virus was isolated from T. atroviride NFCF377 strain and its molecular features were analyzed. The viral genome consists of a single segmented double-stranded RNA and is 9,584 bp in length, with two discontinuous open reading frames (ORF1 and ORF2). A mycoviral structural protein and an RNA-dependent RNA polymerase (RdRp) are encoded by ORF1 and ORF2, respectively, between which is found a canonical shifty heptameric signal motif (AAAAAAC) followed by an RNA pseudoknot. Analysis of sequence similarity and phylogeny showed that it is closely related to members of the proposed family "Fusagraviridae", with a highest similarity to the Trichoderma atroviride mycovirus 1 (TaMV1). Although the sequence similarity of deduced amino acid to TaMV1 was evident, sequence deviations were distinctive at untranslated regions (UTRs) due to the extended size. Thus, we inferred this dsRNA to be a different strain of Trichoderma atroviride mycovirus 1 (TaMV1-NFCF377). Electron microscopy image exhibited an icosahedral viral particle of 40 nm diameter. Virus-cured isogenic isolates were generated and no differences in growth rate, colony morphology, or conidia production were observed between virus-infected and virus-cured strains. However, culture filtrates of TaMV1-NFCF377-infected strain showed enhanced antifungal activity against the plant pathogen Rhizoctonia solani but not to edible mushroom Pleurotus ostreatus. These results suggested that TaMV1-NFCF377 affected the metabolism of the fungal host to potentiate antifungal compounds against a plant pahogen, but this enhanced antifungal activity appeared to be species-specific.

Cell Wall Biogenesis Protein Phosphatase CrSsd1 Is Required for Conidiation, Cell Wall Integrity, and Mycoparasitism in Clonostachys rosea.

Cell wall biogenesis protein phosphatases play important roles in various cellular processes in fungi. However, their functions in the widely distributed mycoparasitic fungus Clonostachys rosea remain unclear, as do their potential for controlling plant fungal diseases. Herein, the function of cell wall biogenesis protein phosphatase CrSsd1 in C. rosea 67-1 was investigated using gene disruption and complementation approaches. The gene-deficient mutant ΔCrSsd1 exhibited much lower conidiation, hyphal growth, mycoparasitic ability, and biocontrol efficacy than the wild-type (WT) strain, and it was more sensitive to sorbitol and Congo red. The results indicate that CrSsd1 is involved in fungal conidiation, osmotic stress adaptation, cell wall integrity, and mycoparasitism in C. rosea.

The Mitogen-Activated Protein Kinase Gene Crmapk Is Involved in Clonostachys chloroleuca Mycoparasitism

Clonostachys chloroleuca is a mycoparasite used for biocontrol of numerous fungal plant pathogens. Sequencing of the transcriptome of C. chloroleuca following mycoparasitization of the sclerotia of Sclerotinia sclerotiorum revealed significant upregulation of a mitogen-activated protein kinase (MAPK)-encoding gene, crmapk. Although MAPKs are known to regulate fungal growth and development, the function of crmapk in C. chloroleuca mycoparasitism is unclear. In this study, we investigated the role of crmapk in C. chloroleuca mycoparasitism through gene knockout and complementation. Deletion of crmapk had no influence on the C. chloroleuca morphological characteristics but could significantly reduce the mycoparasitic ability to sclerotia and biocontrol capacity to soybean Sclerotinia stem rot; crmapk complementation restored these abilities. Transcriptome analysis between Δcrmapk and the wild-type strain revealed numerous genes were significantly down-regulated after crmapk deletion, including cytochrome P450, transporters, and cell wall-degrading enzymes (CWDEs). Our findings indicate that crmapk influences C. chloroleuca mycoparasitism by regulation of genes controlling the activity of CWDEs or antibiotic production. This study provides a basis for further studies of the molecular mechanism of C. chloroleuca mycoparasitism.

Effect of microbial non-volatile metabolites on growth of Fusarium oxysporum f. sp. ciceri and lini

Rhizospheric fungal antagonists activity against phytopathogen by production of volatile and non-volatile metabolites. In vitro assessments revealed their antibiosis and mycoparasitic ability to affect growth of the pathogen. Soil borne fungi, bacteria and actinomycetes were identified and used as antagonistic microbes against some soil borne pathogens. The efficacy of volatile and non-volatile metabolites produced by 14 selected dominant microbes against the common fungal test pathogens (F. oxysporum f. sp. ciceri & lini) were suppressed with colony interaction, volatile, and non-volatile metabolites emanated from different leaf surface microbes like Aspergillus niger, A. terreus, A. luchuensis, Trichoderma harzianum, Penicillium citrinum, Streptomyces rimosus, and S. aureofaciens and prior application of these antagonists effectively reduced the development of Fusarium wilt in chick pea and linseed.

Trichoderma brevicrassum strain TC967 with capacities of diminishing cucumber disease caused by Rhizoctonia solani and promoting plant growth

Some Trichoderma species have been used as biocontrol agents in agriculture. In order to explore additional useful natural resources, a large scale survey of Trichoderma strains from China was conducted and T. brevicrassum strain TC967 was singled out. In vitro assays indicated that it showed high chitinase activity in agar medium, inhibited growth of the plant-pathogenic fungus Rhizoctonia solani (72.14%) in dual culture, and revealed strong mycoparasitic ability through coiling and penetrating hyphae of the pathogen. In vivo assays indicated that it was able to suppress the cucumber disease caused by R. solani to a low disease index (37.5%). Hyphae of strain TC967 colonized cucumber root surface, induced the expression of pathogenesis-related protein genes PR1, PR4 and PR5. In addition, strain TC967 promoted cucumber growth. This is the first report of T. brevicrassum having biocontrol potential and promoting cucumber growth.

Trichoderma harzianum transcriptome in response to cadmium exposure

Cadmium (Cd) is a heavy metal present in the environment mainly as a result of industrial contamination that can cause toxic effects to life. Some microorganisms, as Trichoderma harzianum, a fungus used in biocontrol, are able to survive in polluted environments and act as bioremediators. Aspects about the tolerance to the metal have been widely studied in other fungi although there are a few reports about the response of T. harzianum. In this study, we determined the effects of cadmium over growth of T. harzianum and used RNA-Seq to identify significant genes and processes regulated in the metal presence. Cadmium inhibited the fungus growth proportionally to its concentration although the fungus exhibited tolerance as it continued to grow, even in the highest concentrations used. A total of 3767 (1993 up and 1774 down) and 2986 (1606 up and 1380 down) differentially expressed genes were detected in the mycelium of T. harzianum cultivated in the presence of 1.0 mg mL−1 or 2.0 mg mL−1 of CdCl2, respectively, compared to the absence of the metal. Of these, 2562 were common to both treatments. Biological processes related to cellular homeostasis, transcription initiation, sulfur compound biosynthetic and metabolic processes, RNA processing, protein modification and vesicle-mediated transport were up-regulated. Carbohydrate metabolic processes were down-regulated. Pathway enrichment analysis indicated induction of glutathione and its precursor’s metabolism. Interestingly, it also indicated an intense transcriptional induction, especially by up-regulation of spliceosome components. Carbohydrate metabolism was repressed, especially the mycoparasitism-related genes, suggesting that the mycoparasitic ability of T. harzianum could be affected during cadmium exposure. These results contribute to the advance of the current knowledge about the response of T. harzianum to cadmium exposure and provide significant targets for biotechnological improvement of this fungus as a bioremediator and a biocontrol agent.

Evaluating effective Trichoderma isolates for biocontrol of Rhizoctonia solani causing root rot of Vigna unguiculata

The highly diverse genus Trichoderma has provided many formulations that are alternatives to the chemical pesticides in agriculture. The present study was undertaken to investigate the biocontrol potential of eight Trichoderma species, T. atrobrunneum, T. guizhouense, T. paratroviride, T. pyramidale, T. rufobrunneum, T. simmonsii, T. thermophilum and T. viridulum, against the phytopathogenic fungus Rhizoctonia solani. Trichoderma isolates were first evaluated in vitro by dual culture tests for their antagonism, mycoparasitic ability and antifungal activity against R. solani. Their growth promoting potential was further assessed in relation to phosphate solubilization, indole acetic acid and siderophore production. Five of the isolates were selected and evaluated for their abilities to prompt plant growth and to control R. solani infecting Vigna unguiculata (cowpea) seedlings in vivo. Two most effective isolates, T. guizhouense 9185 and T. simmonsii 8702, significantly (P<0.05) reduced the disease severity incidences (36.6 and 45.0%, respectively) and promoted plant growth, which have good prospects for application.

Comparative analysis of biocontrol agent Trichoderma asperellum ACCC30536 transcriptome during its interaction with Populus davidiana × P. alba var. pyramidalis

After exposure to with Populus davidiana × P. alba var. pyramidalis, the expression of genes in Trichoderma asperellum were compared in four transcriptomes. The top 20 high expression genes included six heat shock proteins and three hydrophobins, indicating that Trichoderma can rapidly adapt to environment stresses and elicit a plant defense response. The genes, involved in the interaction between Trichoderma and plant, showed an increasing expression level, for example sugar transporters, EPL1s, endoxylanases, pectin lyases, and nitrilases. Interestingly, sugar transporters also showed high expression when T. asperellum was cultured on medium lacking a carbon substrate, which would contribute to T. asperellum’s survival and domination in ecological niche competition. And the genes related to mycoparasitism were expressed abundantly following T. asperellum’s interaction with PdPap, indicating the PdPap induction could enhance the mycoparasitic ability of T. asperellum. Twelve chitinases and five glucanases showed higher expression in transcriptome Cs, indicating that T. asperellum secretes both types of enzyme before interacting with pathogens, allowing T. asperellum to implement mycoparasitism and obtain more energy. Many novel transcripts were obtained in each transcriptome, which may play important roles in the biocontrol process of T. asperellum. Interestingly, T. asperellum undergo constitutive alternative splicing in the biocontrol process: Seven biocontrol genes were alternative spliced via intron retention. qRT-PCR analysis proved that intron retention is negatively associated with the expression of chitinase, oligopeptide transporters, and beta-lactamase. However, the percentage of MAPK intron retention was quite low, suggesting that intron retention has little effect on the function of MAPK.

Induction of systemic resistance (ISR) against sheath blight of rice caused by Rhizoctonia solani Kuhn using biological seed treatment with Trichoderma

Sheath blight caused by Rhizoctonia solani Kuhn has recently noticed one of the most important diseases of rice on almost all the high yielding varieties in major rice growing area. In our study morphologically and molecu-larly characterized thirty rhizospheric isolates of Trichoderma (T. harzianum and T. virens) from different locations of Uttarakhand were evaluated for their mycoparasitic ability, disease (sheath blight) suppressing potential and inducing systemic resistance against Rhizoctonia solani. Maximum inhibition in hyphal growth (58.9 %) against R. solani was recorded with isolate PB 2 followed by PB 3 (53.4 %) in confrontation assay. Under glass house con-dition, minimum disease severity (13.6%) was recorded in the treatment where seeds were treated with PB 22 and this treatment also exhibited highest total phenol content (394 ?l/g) 168 hours after pathogen inoculation. Total phenol content was also increased maximally (466 ?l/g) before pathogen inoculation in the treatment where seeds were treated with PB 22. Moreover, high quality ISR activity was recorded with isolates PB 21, 3, 1, 4, 23, 2 and 16 as they reduced more than 34 percent disease and total phenol contents 456 ?l/g, 449 ?l/g, 442 ?l/g, 440 ?l/g and 440 ?l/g, 438 ?l/g and 431 ?l/g were recorded for respective isolates indicated induction of resistance in paddy against sheath blight disease caused by R. solani.

Identification of Cladosporium delicatulum as a mycoparasite of Taphrina pruni

From 2012 until 2014, surveys and frequent visiting the orchards of Hamedan province, west of Iran, have provided strong evidence for the existence of a mycoparasite. The olive green to dark brown velvet colonies, which were observed on infected fruit with Taphrina pruni, Pocket Plum gall, were collected. Morphological characteristics were investigated on Synthetic Nutrient Agar medium. A portion of the translation elongation factor 1-α gene was amplified by using the EF1-526-F and EF1-156-R primers and then sequenced. A mycoparasitic ability was performed by spraying a conidial suspension of Taphrina pruni (1 × 105 spores/mL) and sterile water on leaves. Symptoms appeared in 4–5 days of post-inoculation. The leaves were examined by a light microscope and a scanning electron microscope, and the results of examinations revealed that Cladosporium delicatulum, which had never been reported as a mycoparasite, was in Taphrina pruni.

Mycoparasitic nature of Bionectria sp. strain 6.21

Abstract In this study, a Bionectria sp. strain isolated from citrus rhizosphere was evaluated for its potential in inhibiting the growth of Rhizoctonia solani and Pythium aphanidermatum. It was demonstrated that Bionectria sp. 6.21 inhibited the growth of P. aphanidermatum and R. solani. In dual cultures, however, the antagonist only parasitised R. solani. Regarding the assay involving P. aphanidermatum, a lack of mycoparasitic ability was demonstrated. Crude extract of Bionectria completely inhibited the mycelial growth of both fungi. It appears that the main mechanism involved in the antagonism of Pythium by Bionectria is through antibiotic production. The antagonistic fungus released extracellular secondary metabolites. The metabolites were found to be inhibitory to both plant pathogenic fungi. From the crude extract, eleven fractions were obtained and tested for their antifungal properties. Two of them showed very strong activity against P. aphanidermatum. The obtained results indicated that this biocontrol agent has both antibiotic and mycoparasitic properties. On the other hand, evidence obtained from Scanning Electron Microscopy (SEM) suggests the involvement of an enzymatic process, with enzymatic digestion playing a major role in the parasitism of Bionectria sp. 6.21. In conclusion, these results provide evidence that mainly due to mycoparasitism, this strain has the potential to become a good candidate for biological control.

Trichoderma species mediated differential tolerance against biotic stress of phytopathogens in Cicer arietinum L.

Trichoderma spp. have been reported to aid in imparting biotic as well as abiotic tolerance to plants. However, there are only few reports unfolding the differential ability of separate species of Trichoderma genera generally exploited for their biocontrol potential in this framework. A study was undertaken to evaluate the biocontrol potential of different Trichoderma species namely T. harzianum, T. asperellum, T. koningiopsis, T. longibrachiatum, and T. aureoviride as identified in the group of indigenous isolates from the agricultural soils of Eastern Uttar Pradesh, India. Their biocontrol potential against three major soilborne phytopathogens, i.e., Sclerotium rolfsii, Sclerotinia sclerotiorum, and Colletotrichum capsici was confirmed by dual culture plate technique. Efficient mycoparasitic ability was further assessed in all the isolates in relation to chitinase, β-1,3 glucanase, pectinase, lipase, amylase, and cellulase production while equally consistent results were obtained for their probable phosphate solubilization and indole acetic acid (IAA) production abilities. The selected isolates were further subjected to test their ability to promote plant growth, to reduce disease incidence and to tolerate biotic stress in terms of lignification pattern against S. rolfsii in chickpea plants. Among the identified Trichoderma species, excellent results were observed for T. harzianum and T. koningiopsis indicating better biocontrol potential of these species in the group and thus exhibiting perspective for their commercial exploitation.

Role of gliotoxin in the symbiotic and pathogenic interactions of Trichoderma virens.

Using a gene disruption strategy, we generated mutants in the gliP locus of the plant-beneficial fungus Trichoderma virens that were no longer capable of producing gliotoxin. Phenotypic assays demonstrated that the gliP-disrupted mutants grew faster, were more sensitive to oxidative stress and exhibited a sparse colony edge compared with the WT strain. In a plate confrontation assay, the mutants deficient in gliotoxin production were ineffective as mycoparasites against the oomycete, Pythium ultimum, and the necrotrophic fungal pathogen, Sclerotinia sclerotiorum, but retained mycoparasitic ability against Rhizoctonia solani. Biocontrol assays in soil showed that the mutants were incapable of protecting cotton seedlings from attack by P. ultimum, against which the WT strain was highly effective. The mutants, however, were as effective as the WT strain in protecting cotton seedlings against R. solani. Loss of gliotoxin production also resulted in a reduced ability of the mutants to attack the sclerotia of S. sclerotiorum compared with the WT. The addition of exogenous gliotoxin to the sclerotia colonized by the mutants partially restored their degradative abilities. Interestingly, as in Aspergillus fumigatus, an opportunistic human pathogen, gliotoxin was found to be involved in pathogenicity of T. virens against larvae of the wax moth, Galleria mellonella. The loss of gliotoxin production in T. virens was restored by complementation with the gliP gene from A. fumigatus. We have, thus, demonstrated that the putative gliP cluster of T. virens is responsible for the biosynthesis of gliotoxin, and gliotoxin is involved in mycoparasitism and biocontrol properties of this plant-beneficial fungus.

Histopathological studies of sclerotia of phytopathogenic fungi parasitized by a GFP transformed Trichoderma virens antagonistic strain

The gfp gene from the jellyfish Aequorea victoria, coding for the Green Fluorescent Protein (GFP), was used as a reporter gene to transform a Trichoderma virens strain I10, characterized as having a promising biocontrol activity against a large number of phytopathogenic fungi. On the basis of molecular and biological results, a stable GFP transformant was selected for further experiments. In order to evaluate the effects of GFP transformation on mycoparasitic ability of T. virens I10, sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum and S. minor were inoculated with the T. virens strain I10 GFP transformant or the wild type strain. Statistical analysis of percentages of decayed sclerotia showed that the transformation of the antagonistic isolate with the GFP reporter gene did not modify mycoparasitic activity against sclerotia. Sclerotium colonization was followed by fluorescent microscopy revealing intracellular growth of the antagonist in the cortex ( S. rolfsii) and inter-cellular growth in the medulla ( S. rolfsii, and S. sclerotiorum). The uniformly distributed mycelium of T. virens just beneath the rind of sclerotia of both S. rolfsii and S. sclerotiorum suggests that the sclerotia became infected at numerous randomly distributed locations without any preferential point of entry.

Selection of Verticillium lecanii Isolates with High Potential for Biocontrol of Cucumber Powdery Mildew by Means of Components Analysis at Different Humidity Regimes

To identify characteristics for the selection of Verticillium lecanii isolates with high potential for biocontrol of Sphaerotheca fuliginea under glasshouse conditions, an exploratory study was performed on the effect of water limitation on the development of 14 isolates. The conidial germination, growth and sporulation of isolates of V. lecanii were studied in a tritrophic system on cucumber leaves and in a ditrophic system in Petri dishes. Their mycoparasitic ability was studied in S. fuliginea and Cladosporium cladosporioides . All characteristics were clearly affected by humidity. Four isolates showed good biocontrol potential. The performance of isolates on agar had less predictive value than on powdery mildew. The germination of isolates of V. lecanii was lower and the mycelial growth faster on agar than on mildewed leaves under corresponding humidity conditions. The results suggest that conditions in the phyllosphere differed from the set humidity in the surrounding air. A correlation was found between the lysis of C. cladosporioides growing in dual culture on agar with isolates of V. lecanii and the parasitism of powdery mildew on detached, rooted leaves. C. cladosporioides might offer a suitable substrate for testing isolates of V. lecanii for mycoparasitic potential under various environmental conditions. Conidial germination, growth and sporulation had limited predictive value.

Production of Inter‐ and Intra‐strain Hybrids of Trichoderma spp. by Protoplast Fusion and Evaluation of Their Biocontrol Activity Against Soil‐borne and Foliar Pathogens

AbstractProtoplast fusion techniques were used for the production of new antagonistic strains of Trichoderma spp. Two British and two Italian strains of Trichoderma with different biocontrol potential against Botrytis cinerea, Sclerotinia sclerotiorum and Pythium ultimum, were treated with u.v. radiation and mutants resistant to hygromycin B or propiconzole were selected. Protoplasts were obtained from these mutants after digestion of young hyphae with Novozym 234 and used in inter‐ and intra‐strain protoplast fusion experiments. Hybrids were obtained in 13 crosses isolated on fungicide‐amended media by the inheritance of resistance markers from both parental strains. Selected, fast‐growing, stable mutants were tested in biocontrol trials against P. ultimum on lettuce seedlings and B. cinera on grape bunches in comparison with their parental strains. Intra‐strain hybrids derived from cross XIII were tested in vitro for their mycoparasitic ability on sclerotia of S. sclerotiorum. A high degree of variability in the biocontrol and the mycoparasitic ability of the fusants was observed but no significant increase in the activity was accomplished after fusion, the hybrids being generally less activewas accomplished after fusion, the hybrids being generally less active than their parental strains.