The optimal condition for the production of the artificial fruiting bodies of Paecilomyces japonica in bulk was determined from the observation of the conidial, hyphal, and cultural condition and the medium. The colony was grown 32 mm diameter in 14 days on potato dextrose agar(PDA). Conidia was irregularly long oval-shaped and measured 4.07${\times}$1.56 ${\mu}$m in average. The hyaline hyphae formed transparent bundles which branched out. The fruiting body on the insect surface was measured 30 to 50 mm in length and formed up to 55 branches. Potato dextrose(PD) both was the most effective for the growth of Paecilomyces japonica among the liquid type media. About 3.1 mg mrcelia in dry weight were produced in 50 ml/PD broth. The best condition for the production of conidia under which condition 4.3${\times}$10$^8$ conidia/ml were harvested was pH 5.0 in acidity and 20$^{\circ}C$ following preculture at 24$^{\circ}C$ in temperature for 7 days.
To select efficient entomopathogenic fungal strains of Lecanicillium for the biocontrol of aphid, Myzus persicae, conidial suspension ($1{\times}10\;conidia/ml$) was sprayed onto a detached Chinese cabbage leaf in a petri dish with a dampened filter paper that had 20 nymphs of aphid. Lecancillium strain 4078 and 6543 were the best strains for the biocontrol of aphid at high temperature of $30^{\circ}C$ and low relative humidity (RH) of 85%, respectively. The cumulative mortality of strain 4078 at $30^{\circ}C$ after 3 days was 100% and that of strain 6543 was 90% at 85% RH after 5 days. Strain 4078 also exhibited almost 100% germination ratio of conidia and high rate of mycelial growth at the broad temperature-range of $15{\sim}25^{\circ}C$. The strain 4078 and 6543 were all identified as Lecanicillium species based on the DNA sequences (accession no.: EF026004 and EF026005, respectively) of the ITS regions of the fungi. Excellent production of aerial conidia of strain 6543 was accomplished by using steamed polished rice as the solid culture medium.
The optimum temperature range for conidial germination of Pyriculacia oryzae on a slide glass was $26{\sim}30^{\circ}C$, at which at least four hours of leaf wetness period was required to germinate. Conidial germination was significantly reduced under dry conditions (relative humidity<85%) at $34^{\circ}C$ but not at lower temperature (18, 22, 26, $30^{\circ}C$). Number of lesions developed were greater at $26^{\circ}C$ than at other temperature tested. The average leaf wetness period required for production of a lesion per plant was 22 hours at $18^{\circ}C$, 16 hours at $22^{\circ}C$, 10 hours at $26^{\circ}C$, and 8 hours at $30^{\circ}C$. Less than one lesion per plant occurred at $34^{\circ}C$ even under 24 hours of leaf wetness period. The time period between inoculation and lesion appearance was $7{\sim}8$ days at $18^{\circ}C$, $4{\sim}5$ days at $22^{\circ}C$ and $26^{\circ}C$, and $3{\sim}4$ days at $30^{\circ}C$. The time period required for lesion appearance after inoculation was not affected by leaf wetness period and relative humidity. Lesion length increased most rapidly at $30^{\circ}C$ during the first four days after lesion appearance. Thereater, the rate of increase in lesion length was geratest at $26^{\circ}C$. The average increment of lesion length per day when relative humidity was greater than 90% was 0.7mm at $18^{\circ}C\;and\;22^{\circ}C$, 1mm at $26^{\circ}C$, and 0.8mm at $30^{\circ}C$. When relative humidity was less than 85%, the increments of lesion length per day were approximately $50{\sim}60%$ of those under humid conditions (relative humidity>90%) at all temperature regimes except $30^{\circ}C$. Relative humidity did not significantly affected lesion length at $30^{\circ}C$.
Aspergillus niger IFO 8541 (NRRL 3112) was investigated through a series of UV rays and N-Methyl-N'-Nitro-N-Nitrosoguanidine (NTG) treatments to induce mutants that produce highly active raw starch saccharifying enzyme, and two mutants with strong enzymatic productivity were obtained. The mutants obtained were investigated for their fungal characters, condition of enzyme production, and other activities. Furthermore, the raw starch saccharifying enzyme was purified and the characteristics of purified enzyme were studied. The results obtained were summarized as follows; 1. The color of conidial head of UV-46 mutant obtained from UV rays treatment was changed to tan type and the gelatinated starch saccharifying enzyme productivity and the raw starch saccharifying enzyme productivity increased up to twice and 1.8 times compared to the productivities of original Aspergillus niger IFO 8541 cultured on the wheat bran, respectively. 2. The conidial head color of NG-41 mutant obtained from NTG treatment became lighter than that of parent strain. The gelatinated starch saccharifying enzyme productivity and raw starch saccharifying enzyme productivity increased about 1.8 times, and twice over the Aspergillus niger IFO 8541 parent strain cultured on wheat bran, respectively. The productivity of ${\alpha}$-amylase increased about 3 times more than the parent strain. 3. Two peaks of glucoanlylase and a peak of ${\alpha}$-amylase were obtained when enzyme solution of mutants and parent strain were passed through DEAE-Sephadex A-50 column chromatography. Glucoamylase I showed only gelatinated starch saccharifying enzyme activity. However, glucoamylase II (raw starch saccharifying enzyme) showed both raw starch saccharifying enzyme activity and gelatinated starch saccharifying enzyme activity. 4. Mutant, UV-46 was strengthened in glucoamylase II productivity and mutant NG-41 was strengthened in ${\alpha}$-amylase productivity. 5. Glucoamylase II of mutants and parent strain were appeared to have the same enzymatic properties. 6. Glucoamylase II of mutants and parent strain were recognized as simple enzyme through electrophoresis. 7. The glucoamylase II crystallized showed rhombic board type. 8. The molecular weight, isoelectric point, optimum pH, and optimum temperature of the glucoamylase II crystallized were estimated as 76,000, 3.4, 3.5 and $60^{\circ}C$, respectively.
Interspecific hybrids between Aspergillus niger and Penicillium notatum (Tyr-), hyperlipolytic enzyme-producing fungi, were obtained by nuclear transfer technique. Optimal conditions for formation of intergeneric hybrids were investigated. Maximum production of protoplasts was obtained by 1% Novozyme 234 at $30^{\circ}C$ for 3 hrs and the most effective osmotic stabilizers for the isolation of protoplasts were 0.6 M KCl. Frequencies of hybrid formation by nuclear transfer were $3.8{\times}10^{-3}{\sim}1.3{\times}10^{-3}$. From the observation of genetic stability, conidial size, DNA content, and nuclear stain, it was suggested that their karyotypes are aneuploid. The hybrids showed $1.2{\sim}1.7$ fold higher lipase activities than parental strains. It was strongly supported by results of this study that nuclear transfer technique is much more efficient in the formation of intergeneric hybrids than protoplast fusion and is very useful for the improvement of strains.
Lee, Yong Yoon;Lee, Younmi;Kim, Young Soo;Kim, Hyun Sup;Jeon, Yongho
Research in Plant Disease
/
v.26
no.1
/
pp.8-18
/
2020
Red pepper, one of the major economic crops in Korea, is being affected by anthracnose disease caused by Colletotrichum acutatum. To control this disease, an antagonistic bacterial strain, Bacillus subtilis YGB36 identified by 16S rDNA sequencing, physiological and biochemical analyses is used as a biological control agent. In vitro screening revealed that the strain YGB36 possess strong antifungal activity against the pathogen Cylindrocarpon destructans. The strain exhibited cellulase, protease, amylase, siderophore production and phosphate solubility. In vitro conidial germination of C. acutatum was most drastically inhibited by YGB36 cell suspensions (106 cfu/ml) or culture filtrate. Development of anthracnose symptoms was reduced on detached immature green pepper fruits by treatment with cell suspensions, and its control value was recorded as 65.7%. The YGB36 bacterial suspension treatment enhanced the germination rate of red pepper seeds and promoted root development and growth under greenhouse conditions. The in vitro screening of fungicide and insecticide sensitivity test against YGB36 revealed that the bacterial growth was not affected by any of the insecticides, and 11 fungicides out of 21 used. Collectively, our results clearly suggest that the strain YGB36 is considered as one of the potential biocontrol agents against anthracnose disease in red pepper.
Haejun Jeong;Jonghan Yoon;Hoyoung Park;Min Son;Sook-Young Park;Kwang-Hyung Kim
Research in Plant Disease
/
v.30
no.3
/
pp.219-228
/
2024
Pepper anthracnose, caused by Colletotrichum spp., leads to a decrease in the quantity of pepper fruit production. Molecular diagnosis is crucial for rapid identification of pathogens and determination of fungicide resistance. However, the traditional process of isolating the pathogen, extracting genomic DNA, and analyzing the gene sequence is time-consuming, which delays rapid diagnosis. In this study, we introduced a method using conidia of Colletotrichum spp. instead of genomic DNA, eliminating the need for DNA extraction or special processing for diagnosis. To elucidate this method, sensitivity was assessed through polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) tests using internal transcribed spacer-based primer pairs. Both PCR and qPCR tests showed that detection is feasible with just one conidia, with over 1,000 conidia yielding results comparable to approximately 1 pg of genomic DNA. For amplifying the cytochrome b gene for quinone-outside inhibitor fungicide susceptibility testing, detection from a single conidium is achievable, but a stable PCR product is obtained by increasing the number of cycles to 35. Additionally, the addition of 10% grinding fresh chili pepper paste to V8-Juicea gar medium, which is known for inducing conidia rapidly from the isolates, resulted in 3.2 to 6.0 times more conidia compared to the commonly used potato dextrose agar medium, enhancing the potential for swift testing. Taken together, this study presents a direct utilization of pepper anthracnose conidia through PCR or qPCR, offering a valuable technique for amplifying target genes, such as the minimum conidial amount and barcode genes, for molecular identification of anthracnose disease in pepper through PCR and qPCR analysis.
Sieun Kim;Jong-Hwan Shin;Ha-Kyoung Lee;Soo-hyun Kang;Ji-won Han;Seong-Chan Lee;You-Kyoung Han
Research in Plant Disease
/
v.30
no.3
/
pp.288-293
/
2024
Fusarium basal rot (FBR), caused by the ascomycete fungus Fusarium oxysporum, is an economically important disease of onion worldwide. The most economical and effective way to manage FBR would be the use of FBR resistant onion cultivars. This study was carried out to develop a rapid screening method for resistant onion cultivars in seedling stage. We used the F. oxysporum 19-385 isolate, which causes damping-off in onion seedlings and basal rot in onion bulbs. We optimized broth incubation and medium composition for the production of inoculum, and determined conidial concentration for the preparation of F. oxysporum infected soil. Ten commercial cultivars of onion were evaluated the seedling survival rates and heights by infected soil inoculation methods. As a result, 'K-force' was the most resistant cultivar with 97.4% of relative seedling survival rate against the pathogen, whereas 'Sunpower' was the most susceptible cultivar with 20.0% of relative seedling survival rate.
Aspergillus niger B-15 with strong Endo-polygalacturonase (Endo-PG) activities was selected out from a total of 1,573 fungal strains isolated from various testing materials. A mutant strain, U-46, was obtained from the Aspergillus niger B-15 by repeated irradition of ultra-violet light. The objectives of the study were to investigate the fungal properties of the parental and mutant strains obtained and to study the condition of enzyme production and reaction. The results obtained are summarized as follows: 1. The size of conidial head of the U-46 mutant was smaller than that of the parental strains, B-15 and the length of the conidiophore was also shorter than that of the parental strains. 2. The optimum conditions for the Endo-PG production of the parental B-15 strain in the wheat bran Koji were obtained when 40% of water was added to the wheat bran and the temperature was 30 to $35^{\circ}C$. However, the best condition for the mutant U-46 strain was attained when 60 to 70% of water was added and the temperature was $35^{\circ}C$. The optimum growing periods were two to three days for both parental and mutant strains. 3. Under the optimum producing conditions of each strains, the enzymatic activity of the mutant U-46 was 20 times higher than the Endo-PG of the parental strain, B-15. 4. When both strains were cultured in the wheat bran Koji containing 60% of water at $35^{\circ}C$ for three days, the mutant strain. U-46, was about 46 times higher in the Endo-PG activity and about 18 times greater in Exo-PG activity than the parental strain, B-15. The activities of cellulase, $\alpha$-amylase, and glucoamylase were also highly increased in the mutant strain. 5. The mutant strain, U-46, increased its Endo-PG activity up to 20% over that of ordinary case when 1.2 to 1.5% of ammonium sulphate was added to the wheat bran. 6. The optimum condition for Endo-PG activity of crude enzyme of the mutant strain, U-46, was attained when pH of reaction solution was 4.0 to 4.5 and the temperature was $50^{\circ}C$.
Lee, Mi Rong;Kim, Jong Cheol;Lee, Se Jin;Kim, Sihyeon;Lee, Seok Ju;Park, So Eun;Lee, Wang Hyu;Kim, Jae Su
Korean journal of applied entomology
/
v.56
no.3
/
pp.301-308
/
2017
Locusts, Locusta migratoria (Orthoptera: Acrididae) are periodical unpredictable agricultural pests worldwide and cause serious damage to crop production; however, little consideration has been given to the management of this pest. Herein, we constructed a locust-pathogenic fungal library and confirmed that some fungi could be used as resources for locust management. First, the entomopathogenic fungi were collected from sampled soils using a Tenebrio molitor-based baiting system. For the locust assay, a locust colony was obtained from the National Institute of Agricultural Science and Technology. A total of 34 entomopathogenic fungal granules, which were produced by solid cultures, were placed in the plastic insect-rearing boxes (2 g/box) and nymphs of locust were contained in the box. In 3-7 days, mycosis was observed on the membranous cuticles of the head, abdomen, and legs of locusts. In particular, Metarhizium anisopliae, M. lepidiotae, and Clonostachys rogersoniana exhibited high virulence against the locust. Given that the 34 isolates could be used in field applications, their conidial production and stability (thermotolerance) were further characterized. In the thermotolerance assay, Paecilomyces and Purpureocillium isolates had higher thermotolerance than the other isolates. Most of the fungal isolates produced ca. >$1{\times}10^8conidia/g$ on millet grain medium. In a greenhouse trial, the granular application of M. anisopliae isolate on the soil surface resulted in 85.7% control efficacy. This work suggests that entomopathogenic fungi in a granular form can be effectively used to control the migratory locust.
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