Fungal Biocontrol Research Articles

Metabolite profiling reveals comprehensive effects of Chaetomium globosum on citrus preservation

The huge economic loss of citrus fruit after harvest called for safe and efficient preservatives, as chemically synthesized agents threatened the environment and human health. Herein a biocontrol fungus Chaetomium globosum QY-1 near the orchard in riparian area was identified to have antimicrobial, antioxidant and tyrosinase inhibition activity, which meets the requirements of an ideal preservative. Metabolite profiling based on bioassay-guided fractionation was carried out, and eight polyketones were determined by MS and NMR. The most abundant CheA exhibited strong inhibition to Penicillium digitatum, the main pathogen caused citrus fruit rot. Among these metabolites, Epicoccone and Epicoccolide B showed higher antioxidant activity, while Epicoccone and CheA had higher tyrosinase inhibitory activity. All the activities were close to or even better than the positive control (Vc; glutathione; Vc and arbutin; Bellkute), implying that the metabolites of C. globosum had comprehensive effects as natural preservatives.

Wood pellets as carriers of conidia of Trichoderma atroviride SC1 for soil application

The use of biocontrol agents to control soilborne diseases is a promising alternative to chemical pesticides, however, obtaining a homogeneous distribution and incorporation of conidia of fungal biocontrol agents into the soil is often difficult. Several carriers/formulations have been proposed over time, unfortunately without offering an ultimate solution. We propose the use of wood pellets as a carrier of conidia of a saprophytic fungus that has good biodegradation and biocontrol properties (Trichoderma atroviride SC1). The coating process is based on the direct spraying of wood pellets with a conidial suspension at different rates. Beech, fir, and chestnut wood pellets were compared in terms of relevant physicochemical traits and efficacy in supporting the growth of the fungus. Beech wood pellets displayed the best characteristics in terms of water holding capacity, swelling properties, and disintegration time. T. atroviride SC1 grows best on beech and fir wood pellets and reaches a plateau after nine days of incubation, regardless of the initial coating concentrations. The addition of small quantities of a nitrogen source as tryptone or soy flour, soy proteins, and a mixture of animal proteins used as pet food to the conidial suspension can increase the growth by ten-folds on all types of wood pellets. Our results demonstrate that beech and fir wood pellets could be suitable carriers to deliver and sustain the growth of T. atroviride SC1.

Pathogenicity and Metabolites of Endoparasitic Nematophagous Fungus Drechmeria coniospora YMF1.01759 against Nematodes.

Plant parasitic nematodes cause severe damage to crops. Endoparasitic nematophagous fungi (ENF) are a type of important biocontrol fungi, which can cause disease or kill nematodes by producing various spores. As a major ENF, Drechmeria coniospora displays certain potential for controlling plant-parasitic nematodes. In this study, the pathogenicity and secondary metabolites of the endoparasitic fungus D. coniospora YMF1.01759 were investigated. The strain D. coniospora YMF1.01759 had high infection efficiency against nematodes. The process of infecting nematodes by the strain was observed under an electron microscope. Here, 13 metabolites including one new compound 4(S)-butoxy-3-(butoxymethyl)-2-hydroxycyclopent-2-en-1-one (2) were isolated and identified from the fermentation products of D. coniospora YMF1.01759 cultured in a SDAY solid medium. Furthermore, a bioassay showed that 5-hydroxymethylfuran-2-carboxylic acid (1) is toxic to the root knot nematode Meloidogyne incognita and affects the hatching of its egg. Thereby, the nematicidal mortality attained 81.50% at 100 μg/mL for 48 h. Furthermore, egg hatching was inhibited at the tested concentrations, compared with water control eggs. This is the first report on the secondary metabolites of the ENF D. coniospora. The results indicated that D. coniospora could infect nematodes by spores and produce active metabolites to kill nematodes. The biological control potential of D. coniospora against nematodes was expounded further.

Factors Determining the Variability of Performance of Bio-Control Agents against Root-Knot Nematodes in Vegetable Plants

The application of management strategies against plant-parasitic nematodes (PPNs), an alternative to the use of toxic nematicides, has become of paramount importance due to the recognized environmental impact. Pre-treatments with bio-control agents (BCAs), such as bio-control fungi (BCF, Trichoderma spp.) and arbuscular mycorrhizal fungi (AMF), have been proved to protect many crop plants from endoparasitic sedentary nematodes (ESNs), the most damaging PPN group. However, the use of commercial BCA formulates is not always successful because of an array of variables that influence their performance. One AMF-based and 2 BCF-based commercial formulates were used as soil-drench pre-treatments to protect tomato, egg, and pepper plants from ESN attack. High variability of performance occurred according to the growth stage of treated plants and the amounts of formulates provided per plant. All formulates were highly effective in reducing both root-knot (RKN) and potato cyst (PCN) nematode infection when plants had reached an intermediate growth stage (3.5–5 g plant weight at treatment). However, only specific ranges of doses had to be used. Lower doses were ineffective against nematode attack; higher doses were often toxic to plants. When plants were grown from seeding in BCA-enriched soil, priming against RKNs was even more active. If plants were not challenged by nematodes, BCAs had a low bio-fertilizer effect.

Identification of a new Talaromyces strain DYM25 isolated from the Yap Trench as a biocontrol agent against Fusarium wilt of cucumber

Fusarium equiseti is a pathogenic fungus of plant root rot, and there are few studies on the biocontrol strains of plant wilt caused by F. equiseti. Hence, we conducted a screening and antimicrobial characterization study of marine-origin biocontrol fungi from water samples of the Yap Trench. A new Talaromyces strain DYM25 was screened from water samples of the Yap Trench in the western Pacific Ocean, and its potential as a biocontrol agent against Fusarium wilt of cucumber was studied. 18S rRNA and ITS gene sequencing verified that strain DYM25 belongs to the genus Talaromyces. The growth of F. equiseti was inhibited by strain DYM25 through the antibiosis effect. A preliminary test was first conducted to examine the bioactive stability of filtered DYM25 broth against F. equiseti under various conditions, including high temperature, UV light, alkaline environment, and the presence of metal ions, which indicated its potential as a bio-control agent. The results of the pot experiment showed that F. equiseti caused cucumber wilt, which could be mitigated using the fermentation broth of strain DYM25 (52.9 %). On the other hand, the alkaloid chromogenic reaction showed that the alkaloid salts present in the crude n-butanol extracts were most likely the major components that might have an antimicrobial effect. Therefore, Talaromyces sp. DYM25 represents a new species that can be used as a novel biocontrol agent against cucumber wilt.

Selection of bacteria and fungi for control of soilborne seedling diseases of maize

Plant-based screening experiments were conducted with the aim of identifying biocontrol bacteria and fungi for seed treatment of maize. Candidate microorganisms were evaluated for their protective effects against soilborne infections by species of Fusarium, Globisporangium (syn. Pythium) and Rhizoctonia. The microorganisms tested were bacteria and fungi from maize roots or other sources, including some active microbial components of commercial biocontrol products. Due to the method of isolation chosen, the majority of bacteria from maize roots were spore formers, most of them species of the genera Bacillus,Brevibacillus and Paenibacillus. In pot tests with the potting substrate inoculated with F. culmorum, the level of control provided by seed treatment with the most efficacious bacterial and fungal isolates was comparable or close to the chemical reference seed treatment thiram. The most effective bacteria were species of Pseudomonas, Burkholderia and Streptomyces. Among a subset of approx. 100 bacteria studied, the in vivo and in vitro activities against F.culmorum were only weakly correlated, although some strains deviated from this pattern. The most effective fungi were two strains of Clonostachys rosea and isolates of Trichoderma. The latter and a strain of Gliocladium virens provided also protection against R.solani. Activity against Globisporangium ultimum was recorded for one isolate of Trichoderma and the two strains of C. rosea. A reduction in the impact of seedborne F. culmorum was also observed after seed treatment with two strains of F. oxysporum f. sp. strigae. The results are discussed in relation to previous reports on rhizosphere bacteria of maize and their use in biocontrol of plant pathogens or for plant growth promotion.

Management of root-knot nematode, Meloidogyne incognita and soil borne fungus, Fusarium oxysporum in cucumber using three bioagents under polyhouse conditions.

Complex diseases caused by Meloidogyne incognita and Fusarium fungus in cucumber is the most destructive disease under polyhouses. The experiment was conducted in the polyhouse of the Department of Horticulture, CCS HAU, Hisar, Haryana, India during summer season (2015–16) to evaluate the potential of bacterial and fungal biocontrol agents against Meloidogyne incognita and Fusarium oxysporum f. sp. cucumerinum in cucumber. Bioagents - Trichoderma viride (Tv), Pseudomonas fluorescence (Pf), Purpureocillium lilacinum (Pl) were taken 10 and 20 g kg−1 seed and bioagents liquid formulation, 10- and 15-ml kg−1 seed, were mixed with the potted soil. Chemical as well as untreated check were also maintained. All the treatments significantly improved the plant growth parameter, viz., shoot length (SL), root length (RL), fresh shoot weight (FSW), fresh root weight (FRW), dry shoot weight (DSW) and dry root weight (DRW) as compared to untreated check. However, significant reduction in nematode population and maximum improvement in plant growth parameter was recorded with carbofuran followed by higher dose of bioagents liquid formulation. Among the bioagents, bioagents liquid formulation was most effective in suppressing root knot nematode galling (43 / root system) and final population in soil (131 J2s / 200 cc soil) and fungus wilt incidence (25 %) at 30th day of after germination and significantly improved the plant growth parameters - shoot length (147.3 cm), fresh shoot weight (55.6 g), dry shoot weight (22.51 g) and dry root weight (4.50 g) from other bioagents. Bioagents liquid formulation was effective in suppression of root-knot nematode and fungus complex disease than the powder formulations of bioagents. More studies should be needed in future to evaluate the efficacy of bioagents as seed treatments and soil applications under field conditions.

Current Status and Future Perspective on Enzyme Involving in Biocontrol of Plant Pathogen

To sustain the quality and abundance of fruit, feed and fiber provided by farmers all over the world, plant diseases must be regulated. Plant diseases may be prevented, mitigated, or regulated using a variety of methods. Growers also rely on chemical fertilizers and pesticides for good agronomic and horticultural practices. Such agricultural inputs have taken a vital part in spectacular increases in crop yield and quality over the last 100 years. Microbial enzymes function as biocatalysts for key biochemical reactions and also assist microbes reproduce in a particular niche. The ability of rhizosphere microorganism to increase the growth of plant and control phytopathogens has long been known. Rhizosphere microbes may aid plants in several ways in their fight against phytopathogens. Of all recognized biocontrol pathways, the excretion of lytic enzymes is known as an important way to prevent phytopathogens from living in the region of the rhizosphere. Rhizosphere microorganism produces chitinases, cellulases, proteases, and glucanases in reaction to phytopathogen assault. For assessing antagonist-pathogen interactions, ecological characteristics of antagonists in the rhizosphere, and optimizing the effectiveness of bacterial, fungal, and viral biocontrol agents, new molecular approaches have become available. Given the experience of fungicides in near future, biological management would be another method to control diseases of plant. Since agro-ecosystem is a flexible and functioning structure that involves many variables that affect disease and production of crop, other IPM methods to control diseases of crop are also important in different surrounding conditions. As result, to successfully minimize disease production and crop yield loss in various crop systems, other IPM management mechanisms other than biological control should be considered and implemented.

Regulation of conidiation, polarity growth, and pathogenicity by MrSte12 transcription factor in entomopathogenic fungus, Metarhizium rileyi

Metarhizium rileyi, a well-known filamentous biocontrol fungus, is the main pathogen of numerous field pests, especially noctuid pests. To explore the potential factors involved in the fungal pathogenicity, MrSte12, an important and conserved functional transcription factor in mitogen-activated protein kinase pathway was carried out by functional analysis. Homologous recombination was used to disrupt the MrSte12 gene in M. rileyi. The deletant fungal strain exhibited malformed hyphae and impaired conidiogenesis, and conidia could not be collected from △MrSte12 in vitro towards SMAY medium. Although conidia could be collected again supplemented with KCl within SMAY medium, the conidial germination, growth and stress tolerance were much weaker compared with that in WT. Additionally, △MrSte12 showed a dramatic reduction in virulence in intra-hemolymph injections and no pathogenicity in topical inoculations against noctuid pests, which is due to the failure of appressorium formation. Moreover, the content of chitin and β-1, 3-glucan in cell wall significantly reduced in mutant conidia. These results indicate that the MrSte12 gene markedly contributes to invasive growth and conidiation, as well as the major pathogenicity in M. rileyi.

An Update on Trichoderma Mitogenomes: Complete De Novo Mitochondrial Genome of the Fungal Biocontrol Agent Trichoderma harzianum (Hypocreales, Sordariomycetes), an Ex-Neotype Strain CBS 226.95, and Tracing the Evolutionary Divergences of Mitogenomes in Trichoderma.

Members of the genus Trichoderma (Hypocreales), widely used as biofungicides, biofertilizers, and as model fungi for the industrial production of CAZymes, have actively been studied for the applications of their biological functions. Recently, the study of the nuclear genomes of Trichoderma has expanded in the directions of adaptation and evolution to gain a better understanding of their ecological traits. However, Trichoderma’s mitochondria have received much less attention despite mitochondria being the most necessary element for sustaining cell life. In this study, a mitogenome of the fungus Trichoderma harzianum CBS 226.95 was assembled de novo. A 27,632 bp circular DNA molecule was revealed with specific features, such as the intronless of all core PCGs, one homing endonuclease, and a putative overlapping tRNA, on a closer phylogenetic relationship with T. reesei among hypocrealean fungi. Interestingly, the mitogenome of T. harzianum CBS 226.95 was predicted to have evolved earlier than those of other Trichoderma species and also assumed with a selection pressure in the cox3. Considering the bioavailability, both for the ex-neotype strain of the T. harzianum species complex and the most globally representative commercial fungal biocontrol agent, our results on the T. harzianum CBS 226.95 mitogenome provide crucial information which will be helpful criteria in future studies on Trichoderma.

Identification of microRNA-like RNAs from Trichoderma asperellum DQ-1 during its interaction with tomato roots using bioinformatic analysis and high-throughput sequencing

MicroRNA-like small RNAs (milRNAs) and their regulatory roles in the interaction between plant and fungus have recently aroused keen interest of plant pathologists. Trichoderma spp., one of the widespread biocontrol fungi, can promote plant growth and induce plant disease resistance. To investigate milRNAs potentially involved in the interaction between Trichoderma and tomato roots, a small RNA (sRNA) library expressed during the interaction of T. asperellum DQ-1 and tomato roots was constructed and sequenced using the Illumina HiSeqTM 2500 sequencing platform. From 13,464,142 sRNA reads, we identified 21 milRNA candidates that were similar to other known microRNAs in the miRBase database and 22 novel milRNA candidates that possessed a stable microRNA precursor hairpin structure. Among them, three milRNA candidates showed different expression level in the interaction according to the result of stem-loop RT-PCR indicating that these milRNAs may play a distinct regulatory role in the interaction between Trichoderma and tomato roots. The potential transboundary milRNAs from T. asperellum and their target genes in tomato were predicted by bioinformatics analysis. The results revealed that several interesting proteins involved in plant growth and development, disease resistance, seed maturation, and osmotic stress signal transduction might be regulated by the transboundary milRNAs. To our knowledge, this is the first report of milRNAs taking part in the process of interaction of T. asperellum and tomato roots and associated with plant promotion and disease resistance. The results might be useful to unravel the mechanism of interaction between Trichoderma and tomato.

Dehydration of Ampelomyces quisqualis CPA-9 conidia by adding biodegradable coatings: Biocontrol activity against powdery mildew and physical characterization of the formulated product

Ampelomyces quisqualis CPA-9 was reported as a promising biocontrol fungus against powdery mildew on cucurbits, although its formulation had not been developed. The present work aimed to develop a fluidised-bed spray-dried formulation of CPA-9 that included film-forming compounds, which could improve the behaviour of the microorganism under practical conditions. Film forming compounds, efficacy, shelf life, physical stability as a function of aw, and solubility in water were determined. A film-forming formulation based on native potato starch and pregelatinised potato starch as carrier and binder was obtained, and sucrose plus skimmed milk were used as protective compounds. Dehydrated conidia maintained the efficacy of fresh cells against Podosphaera xanthii on zucchini leaves, and powdery mildew was significantly reduced compared with control treatment. Despite of results obtained for the glassy state guaranteed the stability of the powder against sticking and caking processes, the shelf life of the product was limited to 4 months of storage at 4 °C. On the other hand, the formulation was mainly dispersible, due to the high concentration of native potato starch which reduces the solubility of the product. Results obtained in this work, such as the high efficacy of the developed product, the low cost of the employed substances, the film-forming ability of the rehydrated powder, and finally the effectiveness of the dehydration process, confirmed the potential of the product. Further studies to verify the improvement of CPA-9 behaviour under practical conditions or to improve the storage conditions to enlarge the shelf life of the BCA should be done to consider the developed product as a biocontrol-based product for cucurbits against powdery mildew.

Sanxiapeptin, a linear pentapeptide from Penicillium oxalicum, inhibited the growth of citrus green mold

Penicillium oxalicum has been used as a biocontrol fungus in agriculture for many years, but the antimicrobial substances are still uncertain. Herein, we isolated a linear peptide named Sanxiapeptin in the culture broth of Penicillium oxalicum SG-4 collecting from the Three Gorges riparian zone. Sanxiapeptin exhibited potent inhibitory effect on citrus green mold Penicillium digitatum, the main fungi responsible for postharvest decay. Sanxiapeptin was elucidated as composing of five amino acids, which were β-amino-α-methoxybutyric acid (Amoba), N-Me-l-Thr, d-Thr, N-Me-l-Val and l-Ser. By analyzing three chemically synthesized oligopeptides with similar structures, we found that the first amino acid of Amoba was crucial to the antifungal activity, as was the methylation of peptide bond. Sanxiapeptin may act as an antimicrobial agent by affecting the function of cell membranes or walls. The antimicrobial spectrum, safety and stability analysis supported that Sanxiapeptin was a promising antifungal agent for citrus preservation.

EMPIRICAL STUDY ON LEAFSPOT DISEASE IN MULBERRY PLANT USING BIO CHEMICAL METHODS

Sericulture in India is as old as the ancient Indian culture. In the global scenario, India has emerged as one of the important sericulture practicing countries. The healthy and robust growth of the silkworms depends upon the quality mulberry leaf. Diseases are the major limiting factor in Mulberry cultivation, in which Cercosporamoricola Cooke, the incitant of leaf spot is one among them causing a major damage to the leaf production besides affects the silkworm's health. Four plant extracts and three plant oils tested against the pathogen, Eucalyptus globules at 10% (72.59%) and Madhucaindica oil (3%) evidenced a highest inhibition. Among the different fungal biocontrol agents tested against the pathogen, Trichodermaviride (80.55%) signicantly recorded mycelial growth inhibition over control.

Efficacy of Compost and Some Biocontrol Agents in Controlling Cucumber White Mould Disease under Protected House Conditions

Five different Trichoderma isolates and three different bacterial genera (Serratia marcescens, Bacillus subtilis, Pseudomonas fluorescens) were used in the present study either separately or combined with compost to evaluate their biocontrol potential against Sclerotinia sclerotiorum either in vitro or in vivo. The results showed that all tested bioagents significantly affected the radial growth of the pathogen under laboratory conditions. The highest reduction (61.85%) was detected with isolates T. asperellum followed by T. album which inhibited the radial growth by 60.74%. The combination between T. viride + T. asperellum + T. album was the most effective in reducing disease severity during both seasons and increased yield up to 2.5 kg/plant. The influence of the tested biocontrol fungi and bacteria on essential lysis enzymes activity i.e. chitinase and Beta 1,3- glucanase was investigated. The results showed that both tested fungal and bacterial agents alone or combined with compost affected the activity of both enzymes positively. Similar findings were observed with aspect to three different oxidative enzymes activity i.e. peroxidase (PO), polyphenoloxidase (PPO) and phenylalanine ammonia lyase (PAL). The profiling of peroxidase activity was induced in treated cucumber plants. Moreover, the results revealed that treating cucumber plants with the investigated bioagents expressed new protein bands.

Clonostachys rosea selected by nematicidal screening and its efficacy against Meloidogyne incognita in a greenhouse

ABSTRACT Meloidogyne incognita, one of the most important root-knot nematodes of crops, is still mainly controlled with synthetic agrochemicals. Toward finding eco-friendly alternative controls such as biocontrol fungi or fungal extracts, here fungi were screened in vitro for nematicidal potential, then evaluated for efficacy against M. incognita in greenhouse tomato plants. Forty fungal extracts were also tested at 300 µg mL−1 against second-stage juveniles of M. incognita. Then the strain that produced the most active extract was added (six treatments, mycelium/conidia) to nematode-infested soil in a pot with tomato plants. Agronomic and reproductive variables were measured after 92 days. The best nematicidal extract was from Clonostachys rosea TH27 (LD50 = 375 µg mL−1), which reduced the number of galls, eggs and females in tomato roots and slightly improved agronomic traits compared with untreated plants. For control of M. incognita in pots in protected conditions, 2 g of inoculum per pot (5 kg) is recommended. Our findings confirmed that C. rosea mycelia has potential for control of M. incognita in tomato plants in the greenhouse.

Impact of rhizosphere microorganisms on arsenic (As) transformation and accumulation in a traditional Chinese medical plant.

Panax notoginseng is an important traditional medicinal plant, but the commercial value is threatened by root-rot disease caused by rhizosphere microbes and a potential health risk caused by plant arsenic (As) accumulation. Whether rhizospheric microbes isolated from P. notoginseng rhizosphere soil could impact As uptake and transport into P. notoginseng is not yet known. Among the three root-rot disease-causing pathogens Fusarium flocciferum (PG 1), Fusarium oxysporum (PG 2), and Fusarium solani (PG 3) and one root-rot disease biocontrol fungus Trichoderma koningiopsis (FC 1) and five biocontrol-exerting bacterial species Bacillus siamensis (BC 1), Delftia acidovorans (BC 2), Brevibacillus formosus (BC 3), Mortierella alpine (BC 4), and Bacillus subtilis (BC 5), one As-resistant pathogen and four biocontrol microorganisms with As-resistant ability were identified. The As-transforming ability of the identified fungi and bacteria was ranked in the order of FC 1 > PG 1 and BC 2 > BC 3 > BC 1, respectively. Then, the As-resistant biocontrol and pathogenic microbes were initiated to colonize the rhizosphere of 1-year-old P. notoginseng seedlings growing in artificially As(V)-contaminated soil to evaluate the impact of microbe inoculation on P. notoginseng As uptake and transport capacity. Concentration of As in P. notoginseng tissues decreased in the order of the sequence stem > root > leaf. Compared to treatment without colonization by microorganism, inoculation with microorganisms increased As root uptake efficiency and root As concentration, especially under treatment of inoculation by BC 2 and PG 1 + BC 2. As transport efficiency from root to stem decreased by inoculation with microorganism, especially under treatment with inoculation of BC 2 and PG 1 + BC 2. However, the impact of microorganism colonization on As stem to leaf transport efficiency was not obvious. In summary, inoculation with rhizosphere microbes may increase As accumulation in P. notoginseng root, especially when using bacteria with high As transformation ability. Therefore, it is necessary to evaluate the As transformation capacity before applying biological control microorganism to the rhizosphere of P. notoginseng.

Harzianic Acid from Trichoderma afroharzianum Is a Natural Product Inhibitor of Acetohydroxyacid Synthase.

Acetohydroxyacid synthase (AHAS) is the first enzyme in the branched-chain amino acid biosynthetic pathway and is a validated target for herbicide and fungicide development. Here we report harzianic acid (HA, 1) produced by the biocontrol fungus Trichoderma afroharzianum t-22 (Tht22) as a natural product inhibitor of AHAS. The biosynthetic pathway of HA was elucidated with heterologous reconstitution. Guided by a putative self-resistance enzyme in the genome, HA was biochemically demonstrated to be a selective inhibitor of fungal AHAS, including those from phytopathogenic fungi. In addition, HA can inhibit a common resistant variant of AHAS in which the active site proline is mutated. Structural analysis of AHAS complexed with HA revealed the molecular basis of competitive inhibition, which differs from all known commercial AHAS inhibitors. The alternative binding mode also rationalizes the selectivity of HA, as well as effectiveness toward resistant mutants. A proposed role of HA biosynthesis by Tht22 in the rhizosphere is discussed based on the data.

Thiamine-biosynthesis genes Bbpyr and Bbthi are required for conidial production and cell wall integrity of the entomopathogenic fungus Beauveria bassiana

Beauveria bassiana is an important entomopathogenic fungus used to control a variety of insect pests. Conidia are the infective propagules of the fungus. However, some important factors that influence conidiation are still to be investigated. In this study, a mutant with decreased conidial production and hyphal growth was identified from a random T-DNA insertional library of B. bassiana. The corresponding gene (Bbthi) for this mutation encodes a putative thiazole synthase. Thiazole and pyrimidine are structural components of thiamine (vitamin B1), which is an essential nutrient for all forms of life. Disruption of Bbthi, Bbpyr, a putative pyrimidine synthetic gene, or both in B. bassiana results in a significant decrease of thiamine content. Loss of Bbthi and Bbpyr function significantly decreased the conidial production and hyphal growth, as well as disrupted the integrity of conidial cell wall. However, the defect of Bbpyr and Bbthi does not decrease the virulence of B. bassiana. Our results indicate the importance of thiamine biosynthesis in conidiation of B. bassiana, and provide useful information to produce conidia of entomopathogenic fungi for biocontrol of insect pests.

Inter- and Intra-Continental Genetic Variation in the Generalist Conifer Wood Saprobic Fungus Phlebiopsis gigantea

Phlebiopsis gigantea (Fr.) Jülich is a well-known generalist conifer wood saprobe and a biocontrol fungus used in several world countries to prevent stump infection by tree pathogenic Heterobasidion fungal species. Previous studies have reported the presence of regional and continental genetic differentiation in host-specific fungi, but the presence of such differentiation for generalist wood saprobes such as P. gigantea has not been often studied or demonstrated. Additionally, little information exists on the distribution of this fungus in western North America. The main purposes of this study were: (I) to assess the presence of P. gigantea in California, (II) to explore the genetic variability of P. gigantea at the intra and inter-continental levels and (III) to analyze the phylogeographic relationships between American and European populations. Seven loci (nrITS, ML5–ML6, ATP6, RPB1, RPB2, GPD and TEF1-α) from 26 isolates of P. gigantea from coniferous forests in diverse geographic distribution and from different hosts were analyzed in this study together with 45 GenBank sequences. One hundred seventy-four new sequences were generated using either universal or specific primers designed in this study. The mitochondrial ML5–ML6 DNA and ATP6 regions were highly conserved and did not show differences between any of the isolates. Conversely, DNA sequences from the ITS, RPB1, RPB2, GPD and TEF1-α loci were variable among samples. Maximum likelihood analysis of GPD and TEF1-α strongly supported the presences of two different subgroups within the species but without congruence or geographic partition, suggesting the presence of retained ancestral polymorphisms. RPB1 and RPB2 sequences separated European isolates from American ones, while the GPD locus separated western North American samples from eastern North American ones. This study reports the presence of P. gigantea in California for the first time using DNA-based confirmation and identifies two older genetically distinct subspecific groups, as well as three genetically differentiated lineages within the species: one from Europe, one from eastern North America and one from California, with the latter presumably including individuals from the rest of western North America. The genetic differentiation identified here among P. gigantea individuals from coniferous forests from different world regions indicates that European isolates of this fungus should not be used in North America (or vice versa), and, likewise, commercially available eastern North American P. gigantea isolates should not be used in western North America forests. The reported lack of host specificity of P. gigantea was documented by the field survey and further reinforces the need to only use local isolates of this biocontrol fungus, given that genetically distinct exotic genotypes of a broad generalist microbe may easily spread and permanently alter the microbial biodiversity of native forest ecosystems.