• Research in Plant Disease
• /
• v.28 no.4
• /
• pp.195-203
• /
• 2022

Zea mays, known as maize or corn, is a major staple crop and an important source of energy for humans and animals, thus ensuring global food security. Approximately 9.4% of the loss of total annual corn production is caused by pathogens including fungi, bacteria, and viruses, resulting in economic losses. Although the use of fungicides is one of the most common strategies to control corn diseases, the frequent use of fungicides causes various health problems in humans and animals. In order to overcome this problem, an eco-friendly control strategy has recently emerged as an alternative way. One such eco-friendly control strategy is the use of beneficial microorganisms in the control of plant pathogens. The beneficial microorganisms can control the plant pathogens in various ways, such as spatial competition with plant pathogens, inhibition of fungal or bacterial growth via the production of secondary metabolites or antibiotics, and direct attack to plant pathogens via enzyme activity. Here, we reviewed microorganisms as biocontrol agents against corn diseases.

• 한국균학회소식:학술대회논문집
• /
• 2014.05a
• /
• pp.27-28
• /
• 2014

Beauveria bassiana (Cordycipitaceae, Hypocreales, Ascomycota) is an anamorphic fungus having a potential to be used as a biological control agent because it parasitizes a wide range of arthropod hosts including termites, aphids, beetles and many other insects. A number of bioactive secondary metabolites (SMs) have been isolated from B. bassiana and functionally verified. Among them, beauvericin and bassianolide are cyclic depsipeptides with antibiotic and insecticidal effects belonging to the enniatin family. Non-ribosomal peptide synthetases (NRPSs) play a crucial role in the synthesis of these secondary metabolites. NRPSs are modularly organized multienzyme complexes in which each module is responsible for the elongation of proteinogenic and non-protein amino acids, as well as carboxyl and hydroxyacids. A minimum of three domains are necessary for one NRPS elongation module: an adenylation (A) domain for substrate recognition and activation; a tholation (T) domain that tethers the growing peptide chain and the incoming aminoacyl unit; and a condensation (C) domain to catalyze peptide bond formation. Some of the optional domains include epimerization (E), heterocyclization (Cy) and oxidation (Ox) domains, which may modify the enzyme-bound precursors or intermediates. In the present study, we analyzed genomes of B. bassiana and its allied species in Hypocreales to verify the distribution of NRPS-encoding genes involving biosynthesis of beauvericin and bassianolide, and to unveil the evolutionary processes of the gene clusters. Initially, we retrieved completely or partially assembled genomic sequences of fungal species belonging to Hypocreales from public databases. SM biosynthesizing genes were predicted from the selected genomes using antiSMASH program. Adenylation (A) domains were extracted from the predicted NRPS, NRPS-like and NRPS-PKS hybrid genes, and used them to construct a phylogenetic tree. Based on the preliminary results of SM biosynthetic gene prediction in B. bassiana, we analyzed the conserved gene orders of beauvericin and bassianolide biosynthetic gene clusters among the hypocrealean fungi. Reciprocal best blast hit (RBH) approach was performed to identify the regions orthologous to the biosynthetic gene cluster in the selected fungal genomes. A clear recombination pattern was recognized in the inferred A-domain tree in which A-domains in the 1st and 2nd modules of beauvericin and bassianolide synthetases were grouped in CYCLO and EAS clades, respectively, suggesting that two modules of each synthetase have evolved independently. In addition, inferred topologies were congruent with the species phylogeny of Cordycipitaceae, indicating that the gene fusion event have occurred before the species divergence. Beauvericin and bassianolide synthetases turned out to possess identical domain organization as C-A-T-C-A-NM-T-T-C. We also predicted precursors of beauvericin and bassianolide synthetases based on the extracted signature residues in A-domain core motifs. The result showed that the A-domains in the 1st module of both synthetases select D-2-hydroxyisovalerate (D-Hiv), while A-domains in the 2nd modules specifically activate L-phenylalanine (Phe) in beauvericin synthetase and leucine (Leu) in bassianolide synthetase. antiSMASH ver. 2.0 predicted 15 genes in the beauvericin biosynthetic gene cluster of the B. bassiana genome dispersed across a total length of approximately 50kb. The beauvericin biosynthetic gene cluster contains beauvericin synthetase as well as kivr gene encoding NADPH-dependent ketoisovalerate reductase which is necessary to convert 2-ketoisovalarate to D-Hiv and a gene encoding a putative Gal4-like transcriptional regulator. Our syntenic comparison showed that species in Cordycipitaceae have almost conserved beauvericin biosynthetic gene cluster although the gene order and direction were sometimes variable. It is intriguing that there is no region orthologous to beauvericin synthetase gene in Cordyceps militaris genome. It is likely that beauvericin synthetase was present in common ancestor of Cordycipitaceae but selective gene loss has occurred in several species including C. militaris. Putative bassianolide biosynthetic gene cluster consisted of 16 genes including bassianolide synthetase, cytochrome P450 monooxygenase, and putative Gal4-like transcriptional regulator genes. Our synteny analysis found that only B. bassiana possessed a bassianolide synthetase gene among the studied fungi. This result is consistent with the groupings in A-domain tree in which bassianolide synthetase gene found in B. bassiana was not grouped with NRPS genes predicted in other species. We hypothesized that bassianolide biosynthesizing cluster genes in B. bassiana are possibly acquired by horizontal gene transfer (HGT) from distantly related fungi. The present study showed that B. bassiana is the only species capable of producing both beauvericin and bassianolide. This property led to B. bassiana infect multiple hosts and to be a potential biological control agent against agricultural pests.

• Journal of The Korean Society of Grassland and Forage Science
• /
• v.30 no.1
• /
• pp.77-88
• /
• 2010

In order to ensure good animal health and performance, it is essential to produce forages with high feeding value and good hygienic quality. However, huge amounts of forages consumed by ruminants are contaminated with mold prior to harvest or during storage as hay, straw or silage. These mold can grow in forages only when nutrients are available, correct temperature exist, oxygen is present, and unbound water is available. Fungal 'species can be divided into two groups: field fungi and storage fungi. Field fungi invade the forages while the crop is still in the field, require high moisture conditions, and are such as species of Fusarium, Alternaria, Clodosporium, Diplodia, Gibberrella and Helminthosporium. Storage fungi invade forages during storage and need less moisture than field fungi. These such as species of Aspergillus and Penicillium usually do not occur any problem before harvest. Mold growth can spoil the nutritional aspects of the forages and also results in secondary metabolites that are highly toxic to animal, humans and plants. Moldy feeds are less palatable and may reduce dry matter intake. This, in turn, leads to a reduction of nutrition intake, reducing weight gains or milk production. Performance losses of 5 to 10 percent are typical with moldy feeds. Mycotoxins are toxic substances produced by fungi (molds) growing on crops in the field or storages. While greater than 400 mycotoxins have been chemically identified, the biological or veterinary medical impact of only several mycotoxins is known. Mycotoxins have attracted considerable attention as potential causes for poor performance and health disorders in domestic livestock. They can be carcinogenic, hepatotoxic, hematotoxic, immunosuppressive, estrogenic, or mutagenic. So, feeding moldy forages has adverse effects on animal health and milk consumers. Also, this author reported that rice straw hay was contaminated mycotoxigenic fungi such as Penicillium roqueforti and Fusarium culmorum in Korea. Therefore, it is an urgent need to develop an improved post harvest storage method to reduce nutrient loss and mycotoxin contamination of forages, which will have a positive impact on human health.

• Journal of Food Hygiene and Safety
• /
• v.31 no.3
• /
• pp.194-200
• /
• 2016

Aflatoxins and ochratoxin A (AFTs and OTA) are secondary fungal metabolites produced by several moulds, mainly by Aspergillus flavus by Aspergillus ochraceus and Penicillium verrucosum, and these toxins can be transferred to animals and humans through the ingestion of contaminated feed and food. This study was to develop the analytical method for determination the levels of AFTs ($B_1$, $B_2$, $G_1$ and $G_2$) and OTA in pork. The AFTs and OTA were analyzed simultaneously by electrospray ionization in positive ion mode and mass reaction monitoring (MRM) after solid phase extract (SPE) columns clean-up. Performance characteristics, such as accuracy, precision, linear range, limit of detection (LOD) and quantification (LOQ), were also determined. Matrix-matched standard calibration was used for quantification, obtaining the recoveries in the range of 67.3~108.2% with the relative standard deviations of < 20%. Limits of detection and quantification were also estimated, obtaining the limits of quantification ranged in $0.7{\sim}1.3{\mu}g/kg$. The results of the inter-day study, which was performed with pork samples for 3 days, showed an accuracy of 92.0~109.9%. The precisions (expressed as relative standard deviation values) for the inter day variation were 2.6~17.8%. The method developed in this study was able to carry out the analysis with the satisfactory intensity and accuracy.

• Korean Journal of Microbiology
• /
• v.42 no.2
• /
• pp.83-88
• /
• 2006

This research was performed to determine the production of lovastatin and its HMG-CoA reductase activity produced by fruit bodies and mycelial liquid cultures of domestic edible mushrooms (8 fungal strains). By deter-mining TLC analysis for the confirmation of the presence of lovastatin, all the extracts from fruit bodies and mycelial liquid culture showed same Rf value (0.46), whick was identical to that of the standard lovastatin. In order to extract lovastatin from fruit body, the mixture of water/acetonitrile/methanol was chosen as the most effective solvent. Extracts from fruit body and mycelial liquid culture of pleurotus ostreatus produced the high-est lovastatin 0.98 mg/g based on dry biomass, and 21.90 mg/L, respectively. In the inhibition rate of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, the highest was obtained in P. ostreatus as 67.8% among fruit bodies, and the rates of mycelial liquid culture extracts from P. ostreatus and Laetiporus sulphureus were 37.2% and 29.1%, respectively. Unusually L. sulphureus showed high inhibition rate with low content of lovastatin due to the contribution of campesterol and gamma-sitosterol with hypocholesterolemic activity as metabolites.

• The Plant Pathology Journal
• /
• v.35 no.4
• /
• pp.351-361
• /
• 2019

In our previous study, pyrrolnitrin produced in Pseudomonas chlororaphis G05 plays more critical role in suppression of mycelial growth of some fungal pathogens that cause plant diseases in agriculture. Although some regulators for pyrrolnitrin biosynthesis were identified, the pyrrolnitrin regulation pathway was not fully constructed. During our screening novel regulator candidates, we obtained a white conjugant G05W02 while transposon mutagenesis was carried out between a fusion mutant $G05{\Delta}phz{\Delta}prn::lacZ$ and E. coli S17-1 (pUT/mini-Tn5Kan). By cloning and sequencing of the transposon-flanking DNA fragment, we found that a vfr gene in the conjugant G05W02 was disrupted with mini-Tn5Kan. In one other previous study on P. fluorescens, however, it was reported that the deletion of the vfr caused increased production of pyrrolnitrin and other antifungal metabolites. To confirm its regulatory function, we constructed the vfr-knockout mutant $G05{\Delta}vfr$ and $G05{\Delta}phz{\Delta}prn::lacZ{\Delta}vfr$. By quantifying ${\beta}-galactosidase$ activities, we found that deletion of the vfr decreased the prn operon expression dramatically. Meanwhile, by quantifying pyrrolnitrin production in the mutant $G05{\Delta}vfr$, we found that deficiency of the Vfr caused decreased pyrrolnitrin production. However, production of phenazine-1-carboxylic acid was same to that in the wild-type strain G05. Taken together, Vfr is required for pyrrolnitrin but not for phenazine-1-carboxylic acid biosynthesis in P. chlororaphis G05.

• Korean Journal of Environmental Agriculture
• /
• v.21 no.4
• /
• pp.261-268
• /
• 2002

Biodegradation of monochlorophenols by wood rot fungi such as Daldina concentrica, Trametes versicolor and Pleurotus ostreatus was evaluated by determining their resistance or toxic test and biodegradability. The metabolites of monochlorophenols were also analyzed. Among the three fungi, T. versicolor was the most resistant to 200 ppm of 2-, 3- and 4-chlorophenols, and did not show any inhibitory mycellium growth. But D. concentrica had a little inhibition effect at more than 100 ppm of 3- or 4-chlorophenol. Control cultures of P. ostreatus took even 14 days far the completion of mycellium growth, but the hyphal growth was improved when 2- or 3-chlorophenol were added to the culture. In biodegradation analysis, P. ostreatus showed the highest degradation of 2- or 3-chlorophenol, while T. versicolor was the most effective in 4-chlorophenol. D. concentrica and P. ostreatus slowly degraded 4-chlorophenol. However, T. versicolor had similar degradation capability in the three monochlorophenols, suggesting that the biode- gradation nude is dependent on the fungi as well as the type of monochlorophenol. Several metabolites such as 1,3,5-trihydroxyl benzene, 1-ethyl-1-hydroxyl pentane, 2-propenoicacid, methylmalonic acid and 2-methyl-4-keto-pentan-2-ol were found as products of primary oxidation of 2-, 3- and 4-chlorophenols by intact fungal cultures. fatty acids including tetradecanoic, heptadecanoic and octadecanoic acids were also detected The order of increase of mycellium weight during incubation were P. ostreatus > T. versicolor > D. concentrica. The pH in the culture was not constantly changed depending on incubation days, but the mycellium weight was slightly increased, indicating that the biodegradation of monochlorophenol might have low relationship with the mycellium growth Laccase activities of T. versicolor and P. ostreatus were continuously increased depending on the incubation days, suggesting that the ligninolytic enzyme activity play an important role in the biodegradation of monochlorophenol.

• KSBB Journal
• /
• v.22 no.5
• /
• pp.305-312
• /
• 2007

It is crucial to develop a miniaturized cultivation method for large and rapid screening of high-yielding mutants of monacolin-K, a powerful anti-hypercholesterolemic secondary metabolite biosynthesized by the fungal cells of Monascus ruber. In order to investigate as many strains as possible in a short time, a miniaturized fermentation method especially suitable for the cultivation of the filamentous Monascus mutants was developed using $50m{\ell}$ culture-tube ($7m{\ell}$ of working volume) instead of the traditional $250m{\ell}$ flask ($50m{\ell}$ of working volume). Generally, in filamentous fungal cell fermentations, morphologies in growth and production cultures should be maintained as thick filamentous and compact-pelleted (usually less than 1 mm in diameter) forms, respectively, for enhanced production of secondary metabolites in final production cultures. In this study, we intended to induce the respective optimal morphologies in the miniaturized culture system for the purpose of rapid screening of overproducers. Miniaturized growth culture system was successfully developed due to the mass production of spores in the statistically optimized solid medium. When large amounts of spores were inoculated into the growth cultures, and brown rice flour (20 g/L) was also supplemented to the growth medium, dense filamentous morphologies were successfully induced in the growth cultures performed with the 50 ml culture tubes. It was implied that the amounts of spores inoculated into the growth tube-cultures and the growth medium components should be the key factors for the induction of the filamentous forms in the growth fermentations. Furthermore, in order to statistically optimize production medium, multiple experiments based on Plackett-Burman design and response surface method (RSM) were carried out, resulting in more than 2 fold enhanced production of monacolin-K in the final production cultures with the optimized production medium. Notably, under the production culture conditions with the statistically optimized medium, optimal pellet sizes below 1 mm in diameter were reproducibly induced, in contrast to the thick and viscous filamentous morphologies observed in the previous production cultures.

• Journal of the Society of Cosmetic Scientists of Korea
• /
• v.31 no.4 s.54
• /
• pp.349-357
• /
• 2005

New antioxidative substances for cosmeceuticals were screened from natural resources such as microbial metabolites, mushrooms, and medicinal plants. Four antioxidants were isolated from the fungal metabolite of Eupenicillium shearii and their structures were determined to be new phenolic compounds. The compounds were designated as melanocins A, B, C, and D. Melanocins $A{\sim}D$ exhibited free radical scavenging activity on DPPH and superoxide with $EC_{50}$ values of $21{\sim}94\;and\;7{\sim}84{\mu}M$, respectively, which were stronger activity than those of ${\alpha}-tocopherol$ and BHA. Melanocin A showed anti-wrinkle effects on the UV-irrated hairless mouse skin. A novel hispidin antioxidative compound designated as inoscavin A was isolated from the fruiting body of the mushroom, Inonotus xeranticus. Inoscavin A scavenged superoxide radical with $EC_{50}$ values of $0.03{\mu}g/mL$, and inhibited rat liver microsomal lipid peroxidation with $EC_{50}$ values of $0.3{\mu}g/mL$. Benzastatins $A{\sim}G$, the novel antioxidants isolated from the culture of Streptomyces nitrosporeus showed potent lipid peroxidation inhibitory activity with $EC_{50}$ values of $3{\sim}30{\mu}M$. A cyclopentene compound with strong hypopigmentary effect was isolated from the fungal metabolite of Penicillium sp. and identifed as terrein. Terrein significantly reduced melanin levels in a melanomacyte cell line, Mel-Ab. It showed 10 times stronger activity than kojic acid, but exhibited no cytotoxic effect even in $100{\mu}M$. It was suggested that terrein reduced melanin synthesis by reducing tyrosinase production by MITF down-regulation.

• 한국균학회소식:학술대회논문집
• /
• 2015.05a
• /
• pp.45-46
• /
• 2015

Clubroot disease of crucifers has occurred since 1957. It has spread to the whole China, especially in the southwest and nourtheast where it causes 30-80% loss in some fields. The disease has being expanded in the recent years as seeds are imported and the floating seedling system practices. For its effective control, the Ministry of Agriculture of China set up a program in 2010 and a research team led by Dr. Yueqiu HE, Yunnan Agricultural University. The team includes 20 main reseachers of 11 universities and 5 institutions. After 5 years, the team has made a lot of progresses in disease occurrence regulation, resources collection, resistance identification and breeding, biological agent exploration, formulation, chemicals evaluation, and control strategy. About 1200 collections of local and commercial crucifers were identified in the field and by artificiall inoculation in the laboratories, 10 resistant cultivars were breeded including 7 Chinese cabbages and 3 cabbages. More than 800 antagostic strains were isolated including bacteria, stretomyces and fungi. Around 100 chemicals were evaluated in the field and greenhouse based on its control effect, among them, 6 showed high control effect, especially fluazinam and cyazofamid could control about 80% the disease. However, fluzinam has negative effect on soil microbes. Clubroot disease could not be controlled by bioagents and chemicals once when the pathogen Plasmodiophora brassicae infected its hosts and set up the parasitic relationship. We found the earlier the pathogent infected its host, the severer the disease was. Therefore, early control was the most effective. For Chinese cabbage, all controlling measures should be taken in the early 30 days because the new infection could not cause severe symptom after 30 days of seeding. For example, a biocontrol agent, Bacillus subtilis Strain XF-1 could control the disease 70%-85% averagely when it mixed with seedling substrate and was drenching 3 times after transplanting, i.e. immediately, 7 days, 14 days. XF-1 has been deeply researched in control mechanisms, its genome, and development and application of biocontrol formulate. It could produce antagonistic protein, enzyme, antibiotics and IAA, which promoted rhizogenesis and growth. Its The genome was sequenced by Illumina/Solexa Genome Analyzer to assembled into 20 scaffolds then the gaps between scaffolds were filled by long fragment PCR amplification to obtain complet genmone with 4,061,186 bp in size. The whole genome was found to have 43.8% GC, 108 tandem repeats with an average of 2.65 copies and 84 transposons. The CDSs were predicted as 3,853 in which 112 CDSs were predicted to secondary metabolite biosynthesis, transport and catabolism. Among those, five NRPS/PKS giant gene clusters being responsible for the biosynthesis of polyketide (pksABCDEFHJLMNRS in size 72.9 kb), surfactin(srfABCD, 26.148 kb, bacilysin(bacABCDE 5.903 kb), bacillibactin(dhbABCEF, 11.774 kb) and fengycin(ppsABCDE, 37.799 kb) have high homolgous to fuction confirmed biosynthesis gene in other strain. Moreover, there are many of key regulatory genes for secondary metabolites from XF-1, such as comABPQKX Z, degQ, sfp, yczE, degU, ycxABCD and ywfG. were also predicted. Therefore, XF-1 has potential of biosynthesis for secondary metabolites surfactin, fengycin, bacillibactin, bacilysin and Bacillaene. Thirty two compounds were detected from cell extracts of XF-1 by MALDI-TOF-MS, including one Macrolactin (m/z 441.06), two fusaricidin (m/z 850.493 and 968.515), one circulocin (m/z 852.509), nine surfactin (m/z 1044.656~1102.652), five iturin (m/z 1096.631~1150.57) and forty fengycin (m/z 1449.79~1543.805). The top three compositions types (contening 56.67% of total extract) are surfactin, iturin and fengycin, in which the most abundant is the surfactin type composition 30.37% of total extract and in second place is the fengycin with 23.28% content with rich diversity of chemical structure, and the smallest one is the iturin with 3.02% content. Moreover, the same main compositions were detected in Bacillus sp.355 which is also a good effects biocontol bacterial for controlling the clubroot of crucifer. Wherefore those compounds surfactin, iturin and fengycin maybe the main active compositions of XF-1 against P. brassicae. Twenty one fengycin type compounds were evaluate by LC-ESI-MS/MS with antifungal activities, including fengycin A $C_{16{\sim}C19}$, fengycin B $C_{14{\sim}C17}$, fengycin C $C_{15{\sim}C18}$, fengycin D $C_{15{\sim}C18}$ and fengycin S $C_{15{\sim}C18}$. Furthermore, one novel compound was identified as Dehydroxyfengycin $C_{17}$ according its MS, 1D and 2D NMR spectral data, which molecular weight is 1488.8480 Da and formula $C_{75}H_{116}N_{12}O_{19}$. The fengycin type compounds (FTCPs $250{\mu}g/mL$) were used to treat the resting spores of P. brassicae ($10^7/mL$) by detecting leakage of the cytoplasm components and cell destruction. After 12 h treatment, the absorbencies at 260 nm (A260) and at 280 nm (A280) increased gradually to approaching the maximum of absorbance, accompanying the collapse of P. brassicae resting spores, and nearly no complete cells were observed at 24 h treatment. The results suggested that the cells could be lyzed by the FTCPs of XF-1, and the diversity of FTCPs was mainly attributed to a mechanism of clubroot disease biocontrol. In the five selected medium MOLP, PSA, LB, Landy and LD, the most suitable for growth of strain medium is MOLP, and the least for strains longevity is the Landy sucrose medium. However, the lipopeptide highest yield is in Landy sucrose medium. The lipopeptides in five medium were analyzed with HPLC, and the results showed that lipopeptides component were same, while their contents from B. subtilis XF-1 fermented in five medium were different. We found that it is the lipopeptides content but ingredients of XF-1 could be impacted by medium and lacking of nutrition seems promoting lipopeptides secretion from XF-1. The volatile components with inhibition fungal Cylindrocarpon spp. activity which were collect in sealed vesel were detected with metheds of HS-SPME-GC-MS in eight biocontrol Bacillus species and four positive mutant strains of XF-1 mutagenized with chemical mutagens, respectively. They have same main volatile components including pyrazine, aldehydes, oxazolidinone and sulfide which are composed of 91.62% in XF-1, in which, the most abundant is the pyrazine type composition with 47.03%, and in second place is the aldehydes with 23.84%, and the third place is oxazolidinone with 15.68%, and the smallest ones is the sulfide with 5.07%.