Insect pathogens as biological control agents: Back to the future.

Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic fungal pathogen causing disease in a wide range of plants. This review summarizes current knowledge of mechanisms employed by the fungus to parasitize its host with emphasis on biology, physiology and molecular aspects of pathogenicity. In addition, current tools for research and strategies to combat S. sclerotiorum are discussed. Taxonomy: Sclerotinia sclerotiorum (Lib.) de Bary: kingdom Fungi, phylum Ascomycota, class Discomycetes, order Helotiales, family Sclerotiniaceae, genus Sclerotinia. Identification: Hyphae are hyaline, septate, branched and multinucleate. Mycelium may appear white to tan in culture and in planta. No asexual conidia are produced. Long-term survival is mediated through the sclerotium; a pigmented, multi-hyphal structure that can remain viable over long periods of time under unfavourable conditions for growth. Sclerotia can germinate to produce mycelia or apothecia depending on environmental conditions. Apothecia produce ascospores, which are the primary means of infection in most host plants. Host range: S. sclerotiorum is capable of colonizing over 400 plant species found worldwide. The majority of these species are dicotyledonous, although a number of agriculturally significant monocotyledonous plants are also hosts. Disease symptoms: Leaves usually have water-soaked lesions that expand rapidly and move down the petiole into the stem. Infected stems of some species will first develop dark lesions whereas the initial indication in other hosts is the appearance of water-soaked stem lesions. Lesions usually develop into necrotic tissues that subsequently develop patches of fluffy white mycelium, often with sclerotia, which is the most obvious sign of plants infected with S. sclerotiorum.

Sclerotinia sclerotiorum (Lib.) de Bary: biology and molecular traits of a cosmopolitan pathogen.

Taxonomy: Kingdom Fungi; Phylum Ascomycota; Class Sordariomycetes; Order Hypocreales; Family Nectriaceae; genus Fusarium. Host range: Very broad at the species level. More than 120 different formae speciales have been identified based on specificity to host species belonging to a wide range of plant families. Disease symptoms: Initial symptoms of vascular wilt include vein clearing and leaf epinasty, followed by stunting, yellowing of the lower leaves, progressive wilting, defoliation and, finally, death of the plant. On fungal colonization, the vascular tissue turns brown, which is clearly visible in cross-sections of the stem. Some formae speciales are not primarily vascular pathogens, but cause foot and root rot or bulb rot. Economic importance: Can cause severe losses in many vegetables and flowers, field crops, such as cotton, and plantation crops, such as banana, date palm and oil palm. Control: Use of resistant varieties is the only practical measure for controlling the disease in the field. In glasshouses, soil sterilization can be performed. Useful websites: http://www.broad.mit.edu/annotation/genome/fusarium_group/MultiHome.html; http://www.fgsc.net/Fusarium/fushome.htm; http://www.phi-base.org/query.php

Pathogen profile update: Fusarium oxysporum

Structural and functional genomics investigations are making an important impact on the current understanding and application of microbial agents used for plant disease control. Here, we review the case of Trichoderma spp., the most widely applied biocontrol fungi, which have been extensively studied using a variety of research approaches, including genomics, transcriptomics, proteomics, metabolomics, etc. Known for almost a century for their beneficial effects on plants and the soil, these fungi are the subject of investigations that represent a successful case of translational research, in which 'omics-generated novel understanding is directly translated in to new or improved crop treatments and management methods. We present an overview of the latest discoveries on the Trichoderma expressome and metabolome, of the complex and diverse biotic interactions established in nature by these microbes, and of their proven or potential importance to agriculture and industry.

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Translational Research on Trichoderma: From 'Omics to the Field

Over the past 3 years, the CRISPR/Cas9 system has revolutionized the field of genome engineering. However, its application has not yet been validated in thermophilic fungi. Myceliophthora thermophila, an important thermophilic biomass-degrading fungus, has attracted industrial interest for the production of efficient thermostable enzymes. Genetic manipulation of Myceliophthora is crucial for metabolic engineering and to unravel the mechanism of lignocellulose deconstruction. The lack of a powerful, versatile genome-editing tool has impeded the broader exploitation of M. thermophila in biotechnology. In this study, a CRISPR/Cas9 system for efficient multiplexed genome engineering was successfully developed in the thermophilic species M. thermophila and M. heterothallica. This CRISPR/Cas9 system could efficiently mutate the imported amdS gene in the genome via NHEJ-mediated events. As a proof of principle, the genes of the cellulase production pathway, including cre-1, res-1, gh1-1, and alp-1, were chosen as editing targets. Simultaneous multigene disruptions of up to four of these different loci were accomplished with neomycin selection marker integration via a single transformation using the CRISPR/Cas9 system. Using this genome-engineering tool, multiple strains exhibiting pronounced hyper-cellulase production were generated, in which the extracellular secreted protein and lignocellulase activities were significantly increased (up to 5- and 13-fold, respectively) compared with the parental strain. A genome-wide engineering system for thermophilic fungi was established based on CRISPR/Cas9. Successful expansion of this system without modification to M. heterothallica indicates it has wide adaptability and flexibility for use in other Myceliophthora species. This system could greatly accelerate strain engineering of thermophilic fungi for production of industrial enzymes, such as cellulases as shown in this study and possibly bio-based fuels and chemicals in the future.

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Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering

The study of gene function in filamentous fungi is a field of research that has made great advances in very recent years. A number of transformation and gene manipulation strategies have been developed and applied to a diverse and rapidly expanding list of economically important filamentous fungi and oomycetes. With the significant number of fungal genomes now sequenced or being sequenced, functional genomics promises to uncover a great deal of new information in coming years. This review discusses recent advances that have been made in examining gene function in filamentous fungi and describes the advantages and limitations of the different approaches.

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Approaches to functional genomics in filamentous fungi.

Mycoparasitism of fungal plant pathogens by Trichoderma species is a complex process that in- volves the production and co-ordinated secretion of cell-wall degrading enzymes. Genes implicated in my- coparasitism by Trichoderma atroviride contain motifs in the promoter region, designated MYRE1-MYRE4, that are proposed to act as binding sites for a global inducer of the mycoparasitic response. The aim of our study was to establish whether these motifs also were present in Trichoderma hamatum and whether the presence of these motifs could predict co-expres- sion when T. hamatum was confronted by a patho- gen. Using a combination of targeted, degenerate and inverse PCR, homologues of the mycoparasitism- related genes ech42 (chit42), prb1 and lam1.3 (xbg1.3-110), which encode an endochitinase, pro- teinase, and b-1,3-glucanase, respectively, were cloned and sequenced from T. hamatum. Alignment of the promoter regions of the three genes revealed identical regions in the chit42 and prb1 promoters, which were 6-9 base pairs in length and conserved in position. Specifically, the regulator y motifs MYRE1-MYRE4 were fully conserved, together with a fifth motif, identified by this research. A substrate as- say designed to investigate the response of these genes from T. harzianum and T. hamatum to a simple carbon source (glycerol) showed that, in contrast to chit42 and prb1, xbg1.3-110 was not expressed. Fur- ther comparison of the expression patterns of these three genes between T. harzianum and T. hamatum using the glycerol substrate assay showed that no

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Co-expression of two genes, a chitinase (chit42) and proteinase (prb1), implicated in mycoparasitism by Trichoderma hamatum

Genetic variation of the fungal plant pathogen Sclerotinia sclerotiorum (Lib.) de Bary was examined using DNA fingerprinting. Seventy‐five isolates of S. sclerotiorum were collected from four populations in the South Island of New Zealand. DNA fingerprints were generated for each isolate by Southern blotting using a cloned repetitive sequence, pLK44.20, as a probe. The 47 different fingerprints produced revealed a high level of variation both within and between populations. Comparison of fingerprint similarities indicated substantial local movement of isolates but gave little evidence for long‐range dispersal. A subset of the isolates was tested for mycelial compatibility. Pairs with identical or highly similar DNA fingerprints gave compatible reactions, whereas isolates whose fingerprints were dissimilar were incompatible. The high level of variation observed in New Zealand populations of S. sclerotiorum has relevance for control of diseases caused by this pathogen, as any method of disease cont...

https://www.tandfonline.com/doi/pdf/10.1080/01140671.1999.9514075

Genetic variation in New Zealand populations of the plant pathogen Sclerotinia sclerotiorum

Subtractive hybridisation was used to target novel genes involved in the mycoparasitic interaction of the biocontrol agent Trichoderma hamatum with the phytopathogen Sclerotinia sclerotiorum. Nineteen novel T. hamatum genes were identified that showed increased expression during mycoparasitism compared to a T. hamatum control. Sequence analysis revealed some cDNA fragments had similarity to known fungal or bacterial genes whereas others had no similarity to any genes previously described. Only one of the novel genes has been characterised in another Trichoderma species, the Trichoderma reesei hex1gene. The proteins encoded by the novel genes included three monooxygenases, a metalloendopeptidase, a gluconate dehydrogenase, an endonuclease and a proton ATPase.

Identification of novel Trichoderma hamatum genes expressed during mycoparasitism using subtractive hybridisation

Petri disease causes decline of grapevines worldwide. The grapevine endophyte Phaeomoniella chlamydospora is the most important fungal pathogen associated with this disease. Epidemiological studies of this pathogen have been hampered by its common occurrence in the internal tissue of apparently healthy vines. Development of a molecular marker for a single strain would overcome this limitation and aid experiments designed to answer key questions about the biology of this pathogen. Genetic variation analysis of New Zealand and Italian strains of P. chlamydospora detected a potential molecular marker in New Zealand isolate A21. Characterization of the 1010 bp marker band showed that it had 50% identity to moxY, a gene involved in the aflatoxin biosynthetic pathway of Aspergillus parasiticus. Sequencing of the region flanking the 1010 bp product revealed a single nucleotide polymorphism in the 39 border of the marker band. Primers were designed to amplify a 488 bp fragment encompassing this polymorphic site and cleavage of this product with the restriction enzyme BsrI produced three bands only in isolate A21 and two bands in all other isolates tested. The sensitivity of the PCR-RFLP protocol was increased with a nested PCR approach and the protocol optimized for soil and wood samples. When the nested PCR/RFLP procedure was used to determine the persistence of viable and nonviable spores in soil, the results showed that nonviable spores were un- detected after 8 wk whereas viable spores still could be detected at 17 wk.

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Margaret A. Carpenter

Papers

Approaches to functional genomics in filamentous fungi.

Co-expression of two genes, a chitinase (chit42) and proteinase (prb1), implicated in mycoparasitism by Trichoderma hamatum

Genetic variation in New Zealand populations of the plant pathogen Sclerotinia sclerotiorum

Identification of novel Trichoderma hamatum genes expressed during mycoparasitism using subtractive hybridisation

Development of an isolate-specific marker for tracking Phaeomoniella chlamydospora infection in grapevines