Linocin M18 protein from the insect pathogenic bacterium Brevibacillus laterosporus isolates.

Brevibacillus laterosporus (Bl) is a Gram-positive and spore-forming bacterium. Insect pathogenic strains have been characterised in New Zealand, and two isolates, Bl 1821L and Bl 1951, are under development for use in biopesticides. However, growth in culture is sometimes disrupted, affecting mass production. Based on previous work, it was hypothesised that Tectiviridae phages might be implicated. While investigating the cause of the disrupted growth, electron micrographs of crude lysates showed structural components of putative phages including capsid and tail-like structures. Sucrose density gradient purification yielded a putative self-killing protein of ~30 kDa. N-terminal sequencing of the ~30 kDa protein identified matches to a predicted 25 kDa hypothetical and a 31.4 kDa putative encapsulating protein homologs, with the genes encoding each protein adjacent in the genomes. BLASTp analysis of the homologs of 31.4 kDa amino acid sequences shared 98.6% amino acid identity to the Linocin M18 bacteriocin family protein of Brevibacterium sp. JNUCC-42. Bioinformatic tools including AMPA and CellPPD defined that the bactericidal potential originated from a putative encapsulating protein. Antagonistic activity of the ~30 kDa encapsulating protein of Bl 1821L and Bl 1951during growth in broth exhibited bacterial autolytic activity. LIVE/DEAD staining of Bl 1821L cells after treatment with the ~30 kDa encapsulating protein of Bl 1821L substantiated the findings by showing 58.8% cells with the compromised cell membranes as compared to 37.5% cells in the control. Furthermore, antibacterial activity of the identified proteins of Bl 1821L was validated through gene expression in a Gram-positive bacterium Bacillus subtilis WB800N. KEY POINTS: • Gene encoding the 31.4 kDa antibacterial Linocin M18 protein was identified • It defined the autocidal activity of Linocin M18 (encapsulating) protein • Identified the possible killing mechanism of the encapsulins.

Lysis Cassette-Mediated Exoprotein Release in Yersinia entomophaga Is Controlled by a PhoB-Like Regulator.

Secretion of exoproteins is a key component of bacterial virulence, and is tightly regulated in response to environmental stimuli and host-dependent signals. The entomopathogenic bacterium Yersinia entomophaga MH96 produces a wide range of exoproteins including its main virulence factor, the 2.46 MDa insecticidal Yen-Tc toxin complex. Previously, a high-throughput transposon-based screening assay identified the region of exoprotein release (YeRER) as essential to exoprotein release in MH96. This study defines the role of the YeRER associated ambiguous holin/endolysin-based lysis cluster (ALC) and the novel RoeA regulator in the regulation and release of exoproteins in MH96. A mutation in the ambiguous lysis cassette (ALC) region abolished exoprotein release and caused cell elongation, a phenotype able to be restored through trans-complementation with an intact ALC region. Endogenous ALC did not impact cell growth of the wild type, while artificial expression of an optimized ALC caused cell lysis. Using HolA-sfGFP and Rz1-sfGFP reporters, Rz1 expression was observed in all cells while HolA expression was limited to a small proportion of cells, which increased over time. Transcriptomic assessments found expression of the genes encoding the prominent exoproteins, including the Yen-Tc, was reduced in the roeA mutant and identified a 220 ncRNA of the YeRER intergenic region that, when trans complemented in the wildtype, abolished exoprotein release. A model for Y. entomophaga mediated exoprotein regulation and release is proposed. IMPORTANCE While theoretical models exist, there is not yet any empirical data that links ALC phage-like lysis cassettes with the release of large macro-molecular toxin complexes, such as Yen-Tc in Gram-negative bacteria. In this study, we demonstrate that the novel Y. entomophaga RoeA activates the production of exoproteins (including Yen-Tc) and the ALC at the transcriptional level. The translation of the ALC holin is confined to a subpopulation of cells that then lyse over time, indicative of a complex hierarchical regulatory network. The presence of an orthologous RoeA and a HolA like holin 5' of an eCIS Afp element in Pseudomonas chlororaphis, combined with the presented data, suggests a shared mechanism is required for the release of some large macromolecular protein assemblies, such as the Yen-Tc, and further supports classification of phage-like lysis clusters as type 10 secretion systems.

You are what you eat: fungal metabolites and host plant affect the susceptibility of diamondback moth to entomopathogenic fungi.

Background: Beauveria are entomopathogenic fungi of a broad range of arthropod pests. Many strains of Beauveria have been developed and marketed as biopesticides. Beauveria species are well-suited as the active ingredient within biopesticides because of their ease of mass production, ability to kill a wide range of pest species, consistency in different conditions, and safety with respect to human health. However, the efficacy of these biopesticides can be variable under field conditions. Two under-researched areas, which may limit the deployment of Beauveria-based biopesticides, are the type and amount of insecticidal compounds produced by these fungi and the influence of diet on the susceptibility of specific insect pests to these entomopathogens. Methods: To understand and remedy this weakness, we investigated the effect of insect diet and Beauveria-derived toxins on the susceptibility of diamondback moth larvae to Beauveria infection. Two New Zealand-derived fungal isolates, B. pseudobassiana I12 Damo and B. bassiana CTL20, previously identified with high virulence towards diamondback moth larvae, were selected for this study. Larvae of diamondback moth were fed on four different plant diets, based on different types of Brassicaceae, namely broccoli, cabbage, cauliflower, and radish, before their susceptibility to the two isolates of Beauveria was assessed. A second experiment assessed secondary metabolites produced from three genetically diverse isolates of Beauveria for their virulence towards diamondback moth larvae. Results: Diamondback moth larvae fed on broccoli were more susceptible to infection by B. pseudobassiana while larvae fed on radish were more susceptible to infection by B. bassiana. Furthermore, the supernatant from an isolate of B. pseudobassiana resulted in 55% and 65% mortality for half and full-strength culture filtrates, respectively, while the filtrates from two other Beauveria isolates, including a B. bassiana isolate, killed less than 50% of larvae. This study demonstrated different levels of susceptibility of the insects raised on different plant diets and the potential use of metabolites produced by Beauveria isolates in addition to their conidia.

A single fungal strain was the unexpected cause of a mass aspergillosis outbreak in the world's largest and only flightless parrot.

Kakapo are a critically endangered species of parrots restricted to a few islands off the coast of New Zealand. Kakapo are very closely monitored, especially during nesting seasons. In 2019, during a highly successful nesting season, an outbreak of aspergillosis affected 21 individuals and led to the deaths of 9, leaving a population of only 211 kakapo. In monitoring this outbreak, cultures of aspergillus were grown, and genome sequenced. These sequences demonstrate that, very unusually for an aspergillus outbreak, a single strain of aspergillus caused the outbreak. This strain was found on two islands, but only one had an outbreak of aspergillosis; indicating that the strain was necessary, but not sufficient, to cause disease. Our analysis provides an understanding of the 2019 outbreak and provides potential ways to manage such events in the future.

Genome sequence of the entomopathogenic Serratia entomophila isolate 626 and characterisation of the species specific itaconate degradation pathway.

BACKGROUND: Isolates of Serratia entomophila and S. proteamaculans (Yersiniaceae) cause disease specific to the endemic New Zealand pasture pest, Costelytra giveni (Coleoptera: Scarabaeidae). Previous genomic profiling has shown that S. entomophila isolates appear to have conserved genomes and, where present, conserved plasmids. In the absence of C. giveni larvae, S. entomophila prevalence reduces in the soil over time, suggesting that S. entomophila has formed a host-specific relationship with C. giveni. To help define potential genetic mechanisms driving retention of the chronic disease of S. entomophila, the genome of the isolate 626 was sequenced, enabling the identification of unique chromosomal properties, and defining the gain/loss of accessory virulence factors relevant to pathogenicity to C. giveni larvae. RESULTS: We report the complete sequence of S. entomophila isolate 626, a causal agent of amber disease in C. giveni larvae. The genome of S. entomophila 626 is 5,046,461 bp, with 59.1% G + C content and encoding 4,695 predicted CDS. Comparative analysis with five previously sequenced Serratia species, S. proteamaculans 336X, S. marcescens Db11, S. nematodiphila DH-S01, S. grimesii BXF1, and S. ficaria NBRC 102596, revealed a core of 1,165 genes shared. Further comparisons between S. entomophila 626 and S. proteamaculans 336X revealed fewer predicted phage-like regions and genomic islands in 626, suggesting less horizontally acquired genetic material. Genomic analyses revealed the presence of a four-gene itaconate operon, sharing a similar gene order as the Yersinia pestis ripABC complex. Assessment of a constructed 626::RipC mutant revealed that the operon confer a possible metabolic advantage to S. entomophila in the initial stages of C. giveni infection. CONCLUSIONS: Evidence is presented where, relative to S. proteamaculans 336X, S. entomophila 626 encodes fewer genomic islands and phages, alluding to limited horizontal gene transfer in S. entomophila. Bioassay assessments of a S. entomophila-mutant with a targeted mutation of the itaconate degradation region unique to this species, found the mutant to have a reduced capacity to replicate post challenge of the C. giveni larval host, implicating the itaconate operon in establishment within the host.

Isolation, Purification, and Characterisation of a Phage Tail-Like Bacteriocin from the Insect Pathogenic Bacterium Brevibacillus laterosporus.

The Gram-positive and spore-forming bacterium Brevibacillus laterosporus (Bl) belongs to the Brevibacillus brevis phylogenetic cluster. Isolates of the species have demonstrated pesticidal potency against a wide range of invertebrate pests and plant diseases. Two New Zealand isolates, Bl 1821L and Bl 1951, are under development as biopesticides for control of diamondback moth and other pests. However, due to the often-restricted growth of these endemic isolates, production can be an issue. Based on the previous work, it was hypothesised that the putative phages might be involved. During investigations of the cause of the disrupted growth, electron micrographs of crude lysate of Bl 1821L showed the presence of phages' tail-like structures. A soft agar overlay method with PEG 8000 precipitation was used to differentiate between the antagonistic activity of the putative phage and phage tail-like structures (bacteriocins). Assay tests authenticated the absence of putative phage activity. Using the same method, broad-spectrum antibacterial activity of Bl 1821L lysate against several Gram-positive bacteria was found. SDS-PAGE of sucrose density gradient purified and 10 kD MWCO concentrated lysate showed a prominent protein band of ~48 kD, and transmission electron microscopy revealed the presence of polysheath-like structures. N-terminal sequencing of the ~48 kD protein mapped to a gene with weak predicted amino acid homology to a Bacillus PBSX phage-like element xkdK, the translated product of which shared >90% amino acid similarity to the phage tail-sheath protein of another Bl published genome, LMG15441. Bioinformatic analysis also identified an xkdK homolog in the Bl 1951 genome. However, genome comparison of the region around the xkdK gene between Bl 1821L and Bl 1951 found differences including two glycine rich protein encoding genes which contain imperfect repeats (1700 bp) in Bl 1951, while a putative phage region resides in the analogous Bl 1821L region. Although comparative analysis of the genomic organisation of Bl 1821L and Bl 1951 PBSX-like region with the defective phages PBSX, PBSZ, and PBP 180 of Bacillus subtilis isolates 168 and W23, and Bacillus phage PBP180 revealed low amino acids similarity, the genes encode similar functional proteins in similar arrangements, including phage tail-sheath (XkdK), tail (XkdO), holin (XhlB), and N-acetylmuramoyl-l-alanine (XlyA). AMPA analysis identified a bactericidal stretch of 13 amino acids in the ~48 kD sequenced protein of Bl 1821L. Antagonistic activity of the purified ~48 kD phage tail-like protein in the assays differed remarkably from the crude lysate by causing a decrease of 34.2% in the number of viable cells of Bl 1951, 18 h after treatment as compared to the control. Overall, the identified inducible phage tail-like particle is likely to have implications for the in vitro growth of the insect pathogenic isolate Bl 1821L.

Biological Control of Diamondback Moth-Increased Efficacy with Mixtures of Beauveria Fungi.

Diamondback moth (DBM) is an important horticultural pest worldwide as the larvae of these moths feed on the leaves of cruciferous vegetables. As DBM has developed resistance to more than 100 classes of synthetic insecticides, new biological control options are urgently required. Beauveria species are entomopathogenic fungi recognized as the most important fungal genus for controlling a wide range of agricultural, forestry, and veterinary arthropod pests. Previous research, aimed at developing new Beauveria-based biopesticides for DBM, has focused on screening single isolates of Beauveria bassiana. However, these fungal isolates have individual requirements, which may limit their effectiveness in some environments. This current study separately assessed 14 Beauveria isolates, from a range of habitats and aligned to four different species (Beauveria bassiana, B. caledonica, B. malawiensis, and B. pseudobassiana), to determine the most effective isolate for the control of DBM. Further assays then assessed whether selected combinations of these fungal isolates could increase the overall efficacy against DBM. Six Beauveria isolates (three B. bassiana and three B. pseudobassiana) achieved high DBM mortality at a low application rate with the first documented report of B. pseudobassiana able to kill 100% of DBM larvae. Further research determined that applications of low-virulent Beauveria isolates improved the control of DBM compared to mixtures containing high-virulent isolates. This novel approach increased the DBM pest mortality and shortened the time to kill.

Identification of genes involved in exoprotein release using a high-throughput exoproteome screening assay in Yersinia entomophaga.

Bacterial protein secretion is crucial to the maintenance of viability and pathogenicity. Although many bacterial secretion systems have been identified, the underlying mechanisms regulating their expression are less well explored. Yersinia entomophaga MH96, an entomopathogenic bacterium, releases an abundance of proteins including the Yen-Tc into the growth medium when cultured in Luria Bertani broth at ≤ 25°C. Through the development of a high-throughput exoproteome screening assay (HESA), genes involved in MH96 exoprotein production were identified. Of 4,080 screened transposon mutants, 34 mutants exhibited a decreased exoprotein release, and one mutation located in the intergenic region of the Yen-Tc operon displayed an elevated exoprotein release relative to the wild-type strain MH96. DNA sequencing revealed several transposon insertions clustered in gene regions associated with lipopolysaccharide (LPSI and LPSII), and N-acyl-homoserine lactone synthesis (quorum sensing). Twelve transposon insertions were located within transcriptional regulators or intergenic regions. The HESA will have broad applicability for identifying genes associated with exoproteome production in a range of microorganisms.

Biosynthesis and beneficial effects of microbial gibberellins on crops for sustainable agriculture.

Soil microbes promote plant growth through several mechanisms such as secretion of chemical compounds including plant growth hormones. Among the phytohormones, auxins, ethylene, cytokinins, abscisic acid and gibberellins are the best understood compounds. Gibberellins were first isolated in 1935 from the fungus Gibberella fujikuroi and are synthesized by several soil microbes. The effect of gibberellins on plant growth and development has been studied, as has the biosynthesis pathways, enzymes, genes and their regulation. This review revisits the history of gibberellin research highlighting microbial gibberellins and their effects on plant health with an emphasis on the early discoveries and current advances that can find vital applications in agricultural practices.

Environmental and plant community drivers of plant pathogen composition and richness.

Interactions between individual plant pathogens and their environment have been described many times. However, the relative contribution of different environmental parameters as controls of pathogen communities remains largely unknown. Here we investigate the importance of environmental factors, including geomorphology, climate, land use, soil and plant community composition, for a broad range of aboveground and belowground fungal, oomycete and bacterial plant pathogens. We found that plant community composition is the main driver of the composition and richness of plant pathogens after taking into account all other tested parameters, especially those related to climate and soil. In the face of future changes in climate and land use, our results suggest that changes in plant pathogen community composition and richness will primarily be mediated through changes in plant communities, rather than the direct effects of climate or soils.

Characterization of a new strain of Metarhizium novozealandicum with potential to be developed as a biopesticide.

The fungal species Metarhizium novozealandicum, that occurs only in New Zealand and Australia has been poorly studied.  In this work, a new strain of M. novozealandicum isolated from a larva of Wiseana sp. is described based on morphology, genomic multilocus (ITS, EF-1α and ß-tubulin) phylogeny, growth in different culture media and insecticidal activity. The isolate AgR-F177 was clustered in the same clade with M. novozealandicum. AgR-F177 colonies developed faster on Sabouraud Dextrose Agar (SDA) than on Potato Dextrose Agar (PDA) when incubated at 25°C, with no growth observed at 30°C on either media. Conidia yield on an oat-based medium in semisolid fermentation was 7.41 x 108conidia/g of substrate and a higher yield of 1.68 x 109conidia/g of substrate was obtained using solid fermentation on cooked rice. AgR-F177 formed microsclerotia (MS) in liquid fermentation after 7 days reaching the maximum yield of 3.3 × 103 MS/mL after 10 days. AgR-F177 caused mortality in Wiseana copularis, Costelytra giveni and Plutella xylostella larvae with efficacies up to 100%, 69.2%, and 45.7%, respectively. The ease of production of AgR-F177 with different fermentation systems and its pathogenicity against different insect pests reveal its potential as a new biopesticide.

Development of Plant-Fungal Endophyte Associations to Suppress Phoma Stem Canker in Brassica.

Endophytic microorganisms are found within the tissues of many plants species, with some conferring several benefits to the host plant including resistance to plant diseases. In this study, two putative endophytic fungi that were previously isolated from wild seeds of Brassica, identified as Beauveria bassiana and Pseudogymnoascus pannorum, were inoculated into cultivars of three Brassica species-Brassica napus, Br. rapa and Br. oleracea. Both fungal endophytes were reisolated from above- and below-ground tissues of inoculated plants at four different plant-growth stages, including cotyledon, one-leaf, two-leaf, and four-leaf stages. None of the plants colonised by these fungi exhibited any obvious disease symptoms, indicating the formation of novel mutualistic associations. These novel plant-endophyte associations formed between Brassica plants and Be. bassiana significantly inhibited phoma stem canker, a devastating disease of Brassica crops worldwide, caused by the fungal pathogen Leptosphaeria maculans. The novel association formed with P. pannorum significantly suppressed the amount of disease caused by L. maculans in one out of two experiments. Although biological control is not a new strategy, endophytic fungi with both antiinsect and antifungal activity are a highly conceivable, sustainable option to manage pests and diseases of economically important crops.

Evolution of virulence in a novel family of transmissible mega-plasmids.

Some Serratia entomophila isolates have been successfully exploited in biopesticides due to their ability to cause amber disease in larvae of the Aotearoa (New Zealand) endemic pasture pest, Costelytra giveni. Anti-feeding prophage and ABC toxin complex virulence determinants are encoded by a 153-kb single-copy conjugative plasmid (pADAP; amber disease-associated plasmid). Despite growing understanding of the S. entomophila pADAP model plasmid, little is known about the wider plasmid family. Here, we sequence and analyse mega-plasmids from 50 Serratia isolates that induce variable disease phenotypes in the C. giveni insect host. Mega-plasmids are highly conserved within S. entomophila, but show considerable divergence in Serratia proteamaculans with other variants in S. liquefaciens and S. marcescens, likely reflecting niche adaption. In this study to reconstruct ancestral relationships for a complex mega-plasmid system, strong co-evolution between Serratia species and their plasmids were found. We identify 12 distinct mega-plasmid genotypes, all sharing a conserved gene backbone, but encoding highly variable accessory regions including virulence factors, secondary metabolite biosynthesis, Nitrogen fixation genes and toxin-antitoxin systems. We show that the variable pathogenicity of Serratia isolates is largely caused by presence/absence of virulence clusters on the mega-plasmids, but notably, is augmented by external chromosomally encoded factors.

Mitochondrial evolution in the entomopathogenic fungal genus Beauveria.

Species in the fungal genus Beauveria are pathogens of invertebrates and have been commonly used as the active agent in biopesticides. After many decades with few species described, recent molecular approaches to classification have led to over 25 species now delimited. Little attention has been given to the mitochondrial genomes of Beauveria but better understanding may led to insights into the nature of species and evolution in this important genus. In this study, we sequenced the mitochondrial genomes of four new strains belonging to Beauveria bassiana, Beauveria caledonica and Beauveria malawiensis, and compared them to existing mitochondrial sequences of related fungi. The mitochondrial genomes of Beauveria ranged widely from 28,806 to 44,135 base pairs, with intron insertions accounting for most size variation and up to 39% (B. malawiensis) of the mitochondrial length due to introns in genes. Gene order of the common mitochondrial genes did not vary among the Beauveria sequences, but variation was observed in the number of transfer ribonucleic acid genes. Although phylogenetic analysis using whole mitochondrial genomes showed, unsurprisingly, that B. bassiana isolates were the most closely related to each other, mitochondrial codon usage suggested that some B. bassiana isolates were more similar to B. malawiensis and B. caledonica than the other B. bassiana isolates analyzed.

Auxins of microbial origin and their use in agriculture.

To maintain the world population demand, a sustainable agriculture is needed. Since current global vision is more friendly with the environment, eco-friendly alternatives are desirable. In this sense, plant growth-promoting rhizobacteria could be the choice for the management of soil-borne diseases of crop plants. These rhizobacteria secrete chemical compounds which act as phytohormones. Indole-3-acetic acid (IAA) is the most common plant hormone of the auxin class which regulates various processes of plant growth. IAA compound, in which structure can be found a carboxylic acid attached through a methylene group to the C-3 position of an indole ring, is produced both by plants and microorganisms. Plant growth-promoting rhizobacteria and fungi secrete IAA to promote the plant growth. In this review, IAA production and mechanisms of action by bacteria and fungi along with the metabolic pathways evolved in the IAA secretion and commercial prospects are revised.Key points• Many microorganisms produce auxins which help the plant growth promotion.• These auxins improve the plant growth by several mechanisms.• The auxins are produced through different mechanisms.

A Tale of Two Grass Species: Temperature Affects the Symbiosis of a Mutualistic Epichloë Endophyte in Both Tall Fescue and Perennial Ryegrass.

Many cool-season grasses form permanent, mutualistic symbioses with asexual Epichloë endophytes. These fungal symbionts often perform a protective role within the association as many strains produce secondary metabolites that deter certain mammalian and invertebrate herbivores. Although initially a serious issue for agriculture, due to mammalian toxins that manifested in major animal health issues, selected strains that provide abiotic stress protection to plants with minimal ill effects to livestock are now commercialized and routinely used to enhance pasture performance in many farming systems. These fungal endophytes and their grass hosts have coevolved over millions of years, and it is now generally accepted that most taxonomic groupings of Epichloë are confined to forming compatible associations (i.e., symptomless associations) with related grass genera within a tribe. The most desired compounds associated with Epichloë festucae var. lolii, an endophyte species associated with perennial ryegrass, are peramine and epoxy-janthitrems. No other major secondary metabolites with invertebrate bioactivity have been identified within this association. However, other agriculturally beneficial compounds, such as lolines, have been discovered in related endophyte species that form associations with fescue grasses. A rationale therefore existed to develop novel grass-endophyte associations between loline-producing endophytes originally isolated from tall fescue with elite cultivars of perennial ryegrass to achieve a wider spectrum of insect bioactivity. A suitable loline-producing endophyte strain of Epichloë sp. FaTG-3 was selected and inoculated into perennial ryegrass. We hypothesed that endophyte transmission frequency, endophyte mycelial biomass and endophyte-derived alkaloid production would differ between the original tall fescue host and the artificial association. Consistent with our hypothesis, our data strongly suggest that plant species significantly affected the plant-endophyte association. This effect became more apparent for transmission frequency and endophyte biomass as the plants matured. Overall, the viable endophyte infection frequency was greater in the tall fescue host than in perennial ryegrass, at all sampling dates. Additionally, temperature was found to be a significant factor affecting endophyte transmission frequency, endophyte mycelial biomass and alkaloid production. Implications for the development of novel grass-endophyte associations are discussed.

Phylogenetic determinants of toxin gene distribution in genomes of Brevibacillus laterosporus.

Brevibacillus laterosporus is a globally ubiquitous, spore forming bacterium, strains of which have shown toxic activity against invertebrates and microbes and several have been patented due to their commercial potential. Relatively little is known about this bacterium. Here, we examined the genomes of six published and five newly determined genomes of B. laterosporus, with an emphasis on the relationships between known and putative toxin encoding genes, as well as the phylogenetic relationships between strains. Phylogenetically, strain relationships are similar using average nucleotide identity (ANI) values and multi-gene approaches, although PacBio sequencing revealed multiple copies of the 16S rDNA gene which lessened utility at the strain level. Based on ANI values, the New Zealand isolates were distant from other isolates and may represent a new species. While all of the genomes examined shared some putative toxicity or virulence related proteins, many specific genes were only present in a subset of strains.

Antimicrobial secondary metabolites from agriculturally important bacteria as next-generation pesticides.

The whole organisms can be packaged as biopesticides, but secondary metabolites secreted by microorganisms can also have a wide range of biological activities that either protect the plant against pests and pathogens or act as plant growth promotors which can be beneficial for the agricultural crops. In this review, we have compiled information about the most important secondary metabolites of three important bacterial genera currently used in agriculture pest and disease management.

Lack of involvement of chitinase in direct toxicity of Beauveria bassiana cultures to the aphid Myzus persicae.

The fungal insect pathogen Beauveria bassiana produces a range of insecticidal metabolites and enzymes, including chitinases and proteases, which may assist the disease progression. The enzymes often play a predominant role in the pathogenicity pathway and both chitinases and proteases have previously been shown to be important in host infection. Spray application of supernatants of B. bassiana broth cultures of an isolate from New Zealand caused significant mortality in the green peach aphid, Myzus persicae, within 24 h, demonstrating an apparent contact toxicity. Three-day-old broth cultures were the most effective, with less insect mortality seen using six-day-old broth. However, aphicidal activity increased again when treating aphids with seven-day-old broth. Cultures grew substantially better and produced more potent aphicidal cultures when cultured in media with an initial pH above 5.5. Chitinase was produced a day earlier than the serine protease Pr1, but the peak production periods of these enzymes did not correlate with the aphicidal activities of three- or six-day-old cultures. Cultures treated with EDTA or heated to inactivate the enzymes still showed strong insecticidal activity. Neither beauvericin nor bassianolide, two known insecticidal metabolites, were detected in the supernatants. Therefore the key aphicidal components of B. bassiana cultures were not associated with chitinase nor Pr1 and are yet to be identified.

Antimicrobial secondary metabolites from agriculturally important fungi as next biocontrol agents.

Synthetic chemical pesticides have been used for many years to increase the yield of agricultural crops. However, in the future, this approach is likely to be limited due to negative impacts on human health and the environment. Therefore, studies of the secondary metabolites produced by agriculturally important microorganisms have an important role in improving the quality of the crops entering the human food chain. In this review, we have compiled information about the most important secondary metabolites of fungal species currently used in agriculture pest and disease management.