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Entomopathogenic fungi utilize specific secondary metabolites to defend against insect immunity, thereby enabling colonization of their specific hosts. We are particularly interested in the polyketide synthesis gene pks15, which is involved in metabolite production, and its role in fungal virulence. Targeted disruption of pks15 followed by genetic complementation with a functional copy of the gene would allow for functional characterization of this secondary metabolite biosynthesis gene. Using a Beauveria bassiana ∆pks15 mutant previously disrupted by a bialophos-resistance (bar) cassette, we report here an in-cis complementation at bar cassette using CRISPR/Cas9 gene editing. A bar-specific short guide RNA was used to target and cause a double-strand break in bar, and a donor DNA carrying a wild-type copy of pks15 was co-transformed with the guide RNA. Isolate G6 of ∆pks15 complemented with pks15 was obtained and verified by PCR, Southern analyses and DNA sequencing. Compared to ∆pks15 which showed a marked reduction in sporulation and insect virulence, the complementation in G6 restored with insect virulence, sporulation and conidial germination to wild-type levels. Atomic force and scanning electron microscopy revealed that G6 and wild-type conidial wall surfaces possessed the characteristic rodlet bundles and rough surface while ∆pks15 walls lacked the bundles and were relatively smoother. Conidia of ∆pks15 were larger and more elongated than that of G6 and the wild type, indicating changes in their cell wall organization. Our data indicate that PKS15 and its metabolite are likely not only important for fungal virulence and asexual reproduction, but also cell wall formation. [ABSTRACT FROM AUTHOR]

The goal of this work is to characterize membrane transporter genes in Cercospora fungi required for autoresistance to the photoactivated, active-oxygen-generating toxin cercosporin they produce for infection of host plants. Previous studies implicated a role for diverse membrane transporters in cercosporin resistance. In this study, transporters identified in a subtractive cDNA library between a Cercospora nicotianae wild type and a cercosporin-sensitive mutant were characterized, including two ABC transporters (CnATR2, CnATR3), an MFS transporter (CnMFS2), a uracil transporter, and a zinc transport protein. Phylogenetic analysis showed that only CnATR3 clustered with transporters previously characterized to be involved in cercosporin resistance. Quantitative RT-PCR analysis of gene expression under conditions of cercosporin toxicity, however, showed that only CnATR2 was upregulated, thus this gene was selected for further characterization. Transformation and expression of CnATR2 in the cercosporin-sensitive fungus Neurospora crassa significantly increased cercosporin resistance. Targeted gene disruption of CnATR2 in the wild type C. nicotianae, however, did not decrease resistance. Expression analysis of other transporters in the cnatr2 mutant under conditions of cercosporin toxicity showed significant upregulation of the cercosporin facilitator protein gene ( CFP), encoding an MFS transporter previously characterized as playing an important role in cercosporin autoresistance in Cercospora species. We conclude that cercosporin autoresistance in Cercospora is mediated by multiple genes, and that the fungus compensates for mutations by up-regulation of other resistance genes. CnATR2 may be a useful gene, alone or in addition to other known resistance genes, for engineering Cercospora resistance in crop plants. [ABSTRACT FROM AUTHOR]

Naphthoquinones are deep red polyketide pigments produced by the ant-pathogenic fungus Ophiocordyceps sp. BCC1869. In culture, biosynthesis of these naphthoquinones remains at a low level during the first 20 days and reaches its maximum production level at approximately 50 days. The MFS transporter gene MFS1 was previously identified in Ophiocordyceps sp. BCC1869 from a subtractive EST library between the fungus grown under naphthoquinone-inductive and naphthoquinone-repressive conditions. We cloned and sequenced this transporter gene, which has an open reading frame of 1505 bp and three introns (48, 52, and 58 bp). Phylogenetic analysis showed this MFS transporter was tightly clustered with fungal riboflavin transporters. Functional analysis of this gene was performed by overexpression of MFS1 under the control of a strong, constitutive promoter. We successfully transformed the fungus with this overexpression plasmid using PEG-protoplast transformation, which generated nine transformants per µg of plasmid. RT-PCR indicated that the MFS1 expression level in the overexpressing strains increased 3- to 10-fold compared to the wild type. HPLC analysis of crude extracts of mutants and wild type demonstrated that four naphthoquinone derivatives, erythrostominone, epierythrostominol, deoxyerythrostominone, and deoxyerythrostominol, were the major naphthoquinones produced and excreted in staggering quantities (20- to 2300-fold) in 7-day old liquid cultures by the mutant C7, compared to the wild type. High resolution electrospray ionization mass spectrometry verified mass spectra of these purified metabolites. Three other naphthoquinone derivatives, whose structures have not been identified, were also detected in high amount in the mutant liquid cultures. [ABSTRACT FROM AUTHOR]

Ten entomopathogenic fungi were tested for the control of female adult Thrips palmi Karny in the laboratory. The most virulent fungus Purpureocillium lilacinum CKPL-053, two commercial bioinsecticides and a chemical insecticide were tested for control of T. palmi in two Dendrobium sp. orchid farms. Controls of T. palmi in both farms were highest with P. lilacinum CKPL-053. Purpureocillium lilacinum CKPL-053 and the Odashima strain P. lilacinum produced paecilotoxins A, B, and C when cultured in complete medium 1 but not in potato dextrose broth. Jasmonic acid contents in Dendrobium sp. petals after application of P. lilacinum CKPL-053, B.bassiana CKB-048, M. robertsii CKM-048, and P. lilacinum CKPL-012 spore suspensions were significantly higher than with imidacloprid and water control treatments. This suggests that jasmonic acid might assist in activating plant defense mechanism to exhibit the repellence and/or resistance effect of Dendrobium sp. against T. palmi. The LC and LT for P. lilacinum CKPL-053 against female T. palmi were 2.67 × 10 conidia ml and 2.6 days at the concentration of 1 × 10 conidia ml, respectively. [ABSTRACT FROM AUTHOR]

The population dynamics and in-farm distribution of Thrips palmi Karny were evaluated in two orchid farms, one in the Bangkok metropolitan area and the other in Ratchaburi Province, Thailand. Yellow sticky traps were hung above the orchid canopy (three traps/220 m), and the trapped adults were counted weekly from November 2009 to October 2010 at the first location and from January to December of 2012 at the second location. T. palmi nymphs and adults were both counted weekly on 300 orchid panicles in an area of 2,200 m at both locations. We found that the T. palmi population dynamics at the first location was represented by a W-shaped curve, with the distribution clumped throughout the year, whereas the curve was J-shaped at the second location, with the distribution mostly clumped. Qualitative surveys of thrips species were conducted on ten different orchid genera in ten locations and on 20 different Dendrobium hybrids in 20 locations. Five random sampling plots (1 × 4 m) were examined on each farm. The results indicated that only T. palmi was found on every farm. Moreover, quantitative comparison among five orchid types revealed that Dendrobium flowers with darker color are more attractive to T. palmi. However, this phenomenon does not coincide with Oncidium sp. and Mokara sp. because of their unfavorable morphology for thrips behavior. [ABSTRACT FROM AUTHOR]

Twenty local isolates of entomopathogenic fungi were determined for control of the larvae and adults of Culex quinquefasciatus. In a laboratory experiment, a Penicillium sp. CM-010 caused 100 % mortality of third-instar larvae within 2 h using a conidial suspension of 1 × 10 conidia ml. Its LC was 3 × 10 conidia ml, and the lethal time (LT) was 1.06 h. Cloning and sequencing of its internal transcribed spacer region indicated that this Penicillium species is Penicillium citrinum (100 % identity in 434 bp). Mortality of the adult was highest with Aspergillus flavus CM-011 followed with Metarhizium anisopliae CKM-048 from 1 × 10 conidia ml. P. citrinum CM-010 at 1 × 10 conidia ml killed 100 % larvae within 2 h while Bacillus thuringiensis var. israelensis at 5 ITU ml required 24 h. This P. citrinum CM-010 also greatly reduced survival of C. quinquefasciatus larvae in an unreplicated field test. Light and transmission electron micrographs showed that the fungal conidia were ingested by the larvae and deposited in the gut. The metabolite patulin was produced by P. citrinum CM-010 instead of citrinin. [ABSTRACT FROM AUTHOR]

A correction to the article "Infection and colonization of tissues of the aphid Myzus persicae and cassava mealybug Phenacoccus manihoti by the fungus Beauveria bassiana" that was published in a previous issue of 2013 is presented.

Histopathogenesis of living insects of Myzus persicae Sulzer (Hemiptera: Aphididae) and Phenacoccus manihoti Matile-Ferrero (Hemiptera: Pseudococcidae) by Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) was monitored from penetration through insect death. Important events in aphids included fungal penetration of the integument of the less-resistant leg intersegmental membrane and invasion of natural openings, formation of hyphal bodies in live aphids by three days post-inoculation (PI), and extensive hyphal colonization of the two leg segments closest to the insect body at death of the aphids. Confocal microscopy of green fluorescent protein-labeled B. bassiana in live mealybugs indicated the fungus penetrated the host through the legs and mouthparts. The fungus was scarce in live mealybugs at 1-5 days PI, formed hyphal bodies by six days PI, and growth was limited to parts of dead hosts at 6-7 days PI. In dead mealybugs, hyphal bodies were near solid tissue. Blastospores were in the hemolymph. [ABSTRACT FROM AUTHOR]