Byeongjin Cha

Characterization of HOG1 homologue, CpMK1, from Cryphonectria parasitica and evidence for hypovirus-mediated perturbation of its phosphorylation in response to hypertonic stress

We examined the biological function of cpmk1, which encodes a MAPK of Cryphonectria parasitica, and its regulation by mycovirus. Sequence comparisons revealed that cpmk1 had highest homology with osm1, a hog1‐homologue from Magnaporthe grisea. A growth defect was observed in the cpmk1‐null mutant under hyperosmotic conditions, indicating that cpmk1 functionally belongs to a hog1 subfamily. Immunoblot analyses indicated that the CpMK1 pathway was affected specifically in hyperosmotic conditions by the hypovirus CHV1‐EP713. Moreover, the virus‐infected hypovirulent UEP1 strain also exhibited severe osmosensitivity compared to the virus‐free isogenic strain EP155/2, thus providing additional evidence for viral regulation of cpmk1 in response to a hypertonic stress. Besides osmosensitivity, disruption of cpmk1 resulted in several, but not all, hypovirulence‐associated changes, such as reduced pigmentation, conidiation, laccase production and cryparin expression. However, the cpmk1‐null mutant exhibited an increased accumulation of pheromone gene transcripts. Virulence assays of the cpmk1‐null mutant revealed reduced canker area, but not as severe as that of UEP1. These results suggest that mycoviruses modulate the MAPK and thereby provoke the aberrant expression of target genes, some of which are likely to be implicated in viral symptom development.

Characterization of a fungal protein kinase from Cryphonectria parasitica and its transcriptional upregulation by hypovirus

The chestnut blight fungus Cryphonectria parasitica and its hypovirus comprise useful model system to study the mechanisms of hypoviral infection. We used degenerate primers based on fungal protein kinases to isolate a gene, cppk1, which encodes a novel Ser/Thr protein kinase of C. parasitica. The gene showed highest homology to ptk1, a Ser/Thr protein kinase from Trichoderma reesei. The encoded protein had a predicted mass of 70.5 kDa and a pI of 7.45. Northern blot analyses revealed that the cppk1 transcript was expressed from the beginning of culture, with a slight increase by 5 days of culture. However, its expression was specifically affected by the presence of virus, and it was transcriptionally upregulated in the fungal strain infected with the hypovirus. A kinase assay using Escherichia coli‐derived CpPK1 revealed CpPK1‐specific phosphorylated proteins with estimated masses of 50 kDa and 44 kDa. In addition, the phosphorylation of both proteins was higher in a cell‐free extract from the hypovirulent strain. The increased expression of cppk1 by the introduction of an additional copy results in a subset of viral symptoms of reduced pigmentation and conidiation in a virus‐free isolate. cppk1 overexpression also causes the downregulation of mating factor genes Mf2/1 and Mf2/2, resulting in female sterility. The present study suggests that the hypovirus disturbs fungal signalling by transcriptional upregulation of cppk1, which results in reduced pigmentation and conidiation and female sterility.

A Tannic Acid-Inducible and Hypoviral-Regulated Laccase3 Contributes to the Virulence of the Chestnut Blight Fungus Cryphonectria parasitica

A new laccase gene (lac3) from the chestnut blight fungus Cryphonectria parasitica was induced by the presence of tannic acid, which is abundant in the bark of chestnut trees and is assumed to be one of the major barriers against pathogen infection. However, other commonly known laccase inducers, including ferulic acid, 2,5-xylidine, catechol, and pH, did not induce lac3 transcription. Moreover, the hypovirus modulated the induction of lac3 transcription, abolishing the transcriptional induction of the lac3 gene by tannic acid. A functional analysis of lac3 using a lac3-null mutant indicated that fungal growth and other morphological characteristics, including pigmentation and sporulation, were not affected. However, a virulence assay indicated that the loss of function of a tannic acid-inducible and hypoviral-regulated laccase resulted in reduced virulence without detectable changes in the morphological features. The constitutive expression of lac3 resulted in no significant differences in the necrotic lesions from those caused by the wild type, but its expression in the presence of the hypovirus led to larger lesions than those caused by the hypovirulent strain. These results suggest that the lac3 gene product may not be the only determinant of fungal virulence in chestnut trees but is an important factor.

Potent in Vivo Antifungal Activity against Powdery Mildews of Pregnane Glycosides from the Roots of Cynanchum wilfordii

Two new pregnane glycosides, kidjoranine 3-O-β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 4)-α-L-cymaropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1→4)-α-L-diginopyranosyl-(1 → 4)-β-D-cymaropyranoside (5) and caudatin 3-O-β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 4)-α-L-cymaropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-α-L-diginopyranosyl-(1 → 4)-β-D-cymaropyranoside (6), were isolated from the roots of Cynanchum wilfordii along with four known compounds (1-4). The antifungal activities of the six compounds against barley powdery mildew caused by Blumeria graminis f. sp. hordei were compared to the antifungal activity of polyoxin B. The caudatin glycosides (1, 4, and 6) showed stronger antifungal activities than polyoxin B, whereas kidjoranine glycosides (2, 3, and 5) had weaker activities than polyoxin B. A wettable powder-type formulation (C. wilfordii-WP20) of the ethyl acetate extract from C. wilfordii roots prohibited the development of barley powdery mildew much more effectively than the commercial fungicide polyoxin B-WP10. In addition, C. wilfordii-WP20 effectively controlled strawberry powdery mildew caused by Sphaerotheca humuli under greenhouse conditions. Thus, the crude extract containing the pregnane glycosides can be used as a botanical fungicide for the environmentally benign control of powdery mildews.

Isolation and Identification of Colletotrichum musae from Imported Bananas

Colletotrichum musae was isolated from dark-brown anthracnose lesions on commercial banana (Musa sapientum L.) to establish the causal agent of the symptom. The fungus grew fast and produced white aerial mycelium on PDA. Acervuli developed abundantly on culture plates after incubation for 10 days at . Pinkish conidial masses were produced on the acervuli, which mostly coalesced together, Conidia were aseptate, hyaline, straight, ellipsoid to globose, and 14.56.9 in size. Black, clavate, round, or irregular-shaped appressoria measuring 8.86.8 were readily formed from germ tubes. Setae-like structures were not found either on the lesion or on the cultures. Sclerotia were also absent. Among the media, PDA medium was the best for mycelial growth. The optimum temperature for mycelial growth was , while the optimum pH ranged from pH 5.5 to 6.5. The isolates of C musae caused black necrotic lesions on banana fruits by needle-wound inoculation, and orange-colored spore masses were produced on the lesions. The fungus also caused discoloration on apple fruits inoculated.

Occurrence of diverse dsRNA in a Korean population of the chestnut blight fungus, Cryphonectria parasitica

We analysed 676 isolates from 33 Korean Cryphonectria parasitica subpopulations in Korea for dsRNA incidence and diversity. dsRNA was detected in 84 isolates. Although the dsRNA banding patterns varied in several minor bands, infected isolates could be categorized into two groups. The most common banding pattern occurred in 77 isolates and contained a 12.7-kb band indicative of Cryphonectria hypovirus 1 (CHV1), and several accompanying minor bands with sizes ranging from 0.9-5kb. Northern blot analysis revealed that all 12.7-kb fragments in the dsRNA-containing isolates hybridized to probes corresponding to open reading frames (ORFs) A and B from the reference CHV1 strain (GenBank accession no. M57938). In addition, the sequence of a 1.4-kb cDNA fragment from a representative isolate of the most common group showed 99% sequence similarity to ORF A of CHV1. However, the other group of seven isolates had distinctive bands of 3.5 and 3.3kb, but not the 12.7-kb band. Sequence comparison showed that cloned fragments of these dsRNAs were similar to those of the coat protein and RNA-dependent RNA polymerase genes of chrysovirus, which indicates the occurrence of chrysovirus in the Korean population. Fungal strain identity was assessed via RFLP analysis of the ITS regions. Among the 84 tested isolates, six had different ITS-RFLP patterns (RFLP-II) from that (RFLP-I) of C. parasitica, and are believed to be C. nitschkei, a sympatric species reported on chestnut trees in Japan. The chrysovirus and CHV1 were detected in strains showing both RFLP patterns. However, the chrysovirus was more frequent in the RFLP-II group.

Effect of polyacetylenic acids from Prunella vulgaris on various plant pathogens.

Aims:  This study is aiming at characterizing antifungal substances from the methanol extract of Prunella vulgaris and at investigating those substances’ antifungal and antioomycete activities against various plant pathogens.

Biological Control of Meloidogyne incognita by Aspergillus niger F22 Producing Oxalic Acid

Restricted usage of chemical nematicides has led to development of environmentally safe alternatives. A culture filtrate of Aspergillus niger F22 was highly active against Meloidogyne incognita with marked mortality of second-stage juveniles (J2s) and inhibition of egg hatching. The nematicidal component was identified as oxalic acid by organic acid analysis and gas chromatography-mass spectroscopy (GC-MS). Exposure to 2 mmol/L oxalic acid resulted in 100% juvenile mortality at 1 day after treatment and suppressed egg hatching by 95.6% at 7 days after treatment. Oxalic acid showed similar nematicidal activity against M. hapla, but was not highly toxic to Bursaphelenchus xylophilus. The fungus was incubated on solid medium and dried culture was used for preparation of a wettable powder-type (WP) formulation as an active ingredient. Two WP formulations, F22-WP10 (ai 10%) and oxalic acid-WP8 (ai 8%), were prepared using F22 solid culture and oxalic acid. In a field naturally infested with M. incognita, application of a mixture of F22-WP10 + oxalic acid-WP8 at 1,000- and 500-fold dilutions significantly reduced gall formation on the roots of watermelon plants by 58.8 and 70.7%, respectively, compared to the non-treated control. The disease control efficacy of the mixture of F22-WP10 + oxalic acid-WP8 was significantly higher than that of a chemical nematicide, Sunchungtan (ai 30% fosthiazate). These results suggest that A. niger F22 can be used as a microbial nematicide for the control of root-knot nematode disease.

Nematicidal activities of 4-quinolone alkaloids isolated from the aerial part of Triumfetta grandidens against Meloidogyne incognita.

The methanol extract of the aerial part of Triumfetta grandidens (Tiliaceae) was highly active against Meloidogyne incognita, with second-stage juveniles (J2s) mortality of 100% at 500 μg/mL at 48 h post-exposure. Two 4-quinolone alkaloids, waltherione E (1), a new alkaloid, and waltherione A (2), were isolated and identified as nematicidal compounds through bioassay-guided fractionation and instrumental analysis. The nematicidal activities of the isolated compounds against M. incognita were evaluated on the basis of mortality and effect on egg hatching. Compounds 1 and 2 exhibited high mortalities against J2s of M. incognita, with EC50 values of 0.09 and 0.27 μg/mL at 48 h, respectively. Compounds 1 and 2 also exhibited a considerable inhibitory effect on egg hatching, which inhibited 91.9 and 87.4% of egg hatching, respectively, after 7 days of exposure at a concentration of 1.25 μg/mL. The biological activities of the two 4-quinolone alkaloids were comparable to those of abamectin. In addition, pot experiments using the crude extract of the aerial part of T. grandidens showed that it completely suppressed the formation of gall on roots of plants at a concentration of 1000 μg/mL. These results suggest that T. grandidens and its bioactive 4-quinolone alkaloids can be used as a potent botanical nematicide in organic agriculture.

Antifungal Activity of Benzoic Acid from Bacillus subtilis GDYA-1 against Fungal Phytopathogens

A bacterial strain antagonistic to some fungal phytopathogens was isolated from the stem of a Persimmon tree in Yeongam, Korea. This bacterium was identified as Bacillus subtilis by 16S rRNA gene sequencing and designated as B. subtilis GDYA-1. In in vivo experiment, the fermentation broth exhibited antifungal activities against Magnaporthe oryzae on rice plants, Phytophthora infestans on tomato plants, and Puccinia recondita on wheat plants. We isolated one antifungal compound and its chemical structure was determined by mass and H-NMR spectral data. The antifungal substance was identified as benzoic acid. It inhibited mycelial growth of M. oryzae, Rhizoctonia solani, Sclerotinia sclerotiorum, and P. capsici with minimum inhibition concentration (MIC) values, ranging from 62.5 to 125 µg/ml. Moreover, the substance effectively suppressed Phytophthora blight of red pepper caused by P. capsici in a pot experiment. To the author’s knowledge, this is the first report on the antifungal activity of benzoic acid against phytopathogenic fungi. Benzoic acid and B. subtilis GDYA-1 may contribute to environmental-friendly protect crops from phytopathogenic fungi.