In vitro studies of an important medicinal plant, Swertia chirata Buch. -Hams. Ex wall. And its biological activities

Balaraju Kotnala,Kyung Seok Park 한국국제농업개발학회 2010 한국국제농업개발학회 추계학술발표논문집 Vol.2010 No.-

Paromomycin Derived from Streptomyces sp. AG-P 1441 Induces Resistance against Two Major Pathogens of Chili Pepper

( Kotnala Balaraju ),( Chang Jin Kim ),( Dong Jin Park ),( Ki Woong Nam ),( Kecheng Zhang ),( Mee Kyung Sang ),( Kyungseok Park ) 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.9

This is the first report that paromomycin, an antibiotic derived from Streptomyces sp. AG-P 1441 (AG-P 1441), controlled Phytophthora blight and soft rot diseases caused by Phytophthora capsici and Pectobacterium carotovorum, respectively, in chili pepper (Capsicum annum L.). Chili pepper plants treated with paromomycin by foliar spray or soil drenching 7 days prior to inoculation with P. capsici zoospores showed significant (p < 0.05) reduction in disease severity (%) when compared with untreated control plants. The disease severity of Phytophthora blight was recorded as 8% and 50% for foliar spray and soil drench, respectively, at 1.0 ppm of paromomycin, compared with untreated control, where disease severity was 83% and 100% by foliar spray and soil drench, respectively. A greater reduction of soft rot lesion areas per leaf disk was observed in treated plants using paromomycin (1.0 μg/ml) by infiltration or soil drench in comparison with untreated control plants. Paromomycin treatment did not negatively affect the growth of chili pepper. Furthermore, the treatment slightly promoted growth; this growth was supported by increased chlorophyll content in paromomycin-treated chili pepper plants. Additionally, paromomycin likely induced resistance as confirmed by the expression of pathogenesis-related (PR) genes: PR-1, β-1,3-glucanase, chitinase, PR-4, peroxidase, and PR-10, which enhanced plant defense against P. capsici in chili pepper. This finding indicates that AG-P 1441 plays a role in pathogen resistance upon the activation of defense genes, by secretion of the plant resistance elicitor, paromomycin.

Identification and Characterization of Diplodia parva and Diplodia crataegicola Causing Black Rot of Chinese Quince

Sungmun Kwon(Sungmun Kwon),Jungyeon Kim(Jungyeon Kim),Younmi Lee(Younmi Lee),Kotnala Balaraju(Kotnala Balaraju ),Yongho Jeon(Yongho Jeon) 한국식물병리학회 2023 Plant Pathology Journal Vol.39 No.3

Fungal isolates from infected Chinese quince trees were found to cause black rot in Yeongcheon, Gyeong-sangbuk Province, Korea. The quince leaves withered and turned reddish-brown and fruits underwent black mummification. To elucidate the cause of these symp-toms, the pathogen was isolated from infected leaf and fruit tissues on potato dextrose agar and Levan media. Several fungal colonies forming a fluffy white or dark gray mycelium and two types of fungi forming an aerial white mycelium, growing widely at the edges, were iso-lated. Microscopic observations, investigation of fungal growth characteristics on various media, and molecu-lar identification using an internal transcribed spacer, β-tubulin, and translation elongation factor 1-α genes were performed. The fungal pathogens were identified as Diplodia parva and Diplodia crataegicola. Pathogenic-ity tests revealed that the pathogen-inoculated fruits ex-hibited a layered pattern, turning brown rotting; leaves showed circular brown necrotic lesions. The developed symptoms were similar to those observed in the field. Fungal pathogens were reisolated to fulfill Koch’s pos-tulates. Apples were inoculated with fungal pathogens to investigate the host range. Strong pathogenicity was evident in the fruits, with browning and rotting symp-toms 3 days after inoculation. To determine pathogen control, a fungicidal sensitivity test was conducted us-ing four registered fungicides. Thiophanate-methyl, propineb, and tebuconazole inhibited the mycelial growth of pathogens. To the best of our knowledge, this is the first report on the isolation and identification of the fungal pathogens D. parva and D. crataegicola from infected fruits and leaves of Chinese quince, causing black rot disease in Korea.

Cryopreservation of in vitro Grown Shoot Tips of Strawberry (Fragaria × ananassa Duch.) Genetic Resources by Droplet-vitrification

이영이,Kotnala Balaraju,송재영,이정윤,이선이,이정로,윤문섭,김행훈 한국자원식물학회 2019 한국자원식물학회지 Vol.32 No.6

This study describes an efficient and widely applicable droplet-vitrification following cryopreservation for shoot tips of strawberry (Fragaria × ananassa Duch.) cvs. ‘Wonkyo3114’ and ‘Gurumi40’. The shoot tips were precultured in Murashige and Skoog (MS) liquid medium supplemented with sucrose (0.3-0.5M). Precultured explants were osmoprotected with loading solution (LS, C4) containing 20% glycerol and 20% sucrose for 40 min and exposed to dehydration solution (B5) containing 40% glycerol and 40% sucrose for 40 min at 25℃, Subsequently, the explants were transferred onto droplets containing 2.5 µL PVS3 on sterilized aluminum foils (4 ㎝ × 0.5 ㎝) prior to direct immersion in liquid nitrogen (LN) for 1 h. The highest regrowth rate (%) in both the cultivars was obtained when the shoot tips were precultured with MS + 0.3M sucrose for 40 h at 25℃. The cryopreserved shoots tips exhibited 55% regrowth rate by culturing in NH4NO3-free MS medium supplemented with 3% sucrose, 1.0 g/L casein, 1.0㎎/L GA3, and 0.5 ㎎/L BA for 5 weeks and in MS medium supplemented with 0.5 ㎎/L GA3 for 8 weeks. This result shows that droplet-vitrification could be employed as a promising method for cryostorage of strawberry germplasm.

Biological Control of Apple Anthracnose by Paenibacillus polymyxa APEC128, an Antagonistic Rhizobacterium

김영수,Kotnala Balaraju,전용호 한국식물병리학회 2016 Plant Pathology Journal Vol.32 No.3

The present study investigated the suppression of the disease development of anthracnose caused by Colleto-trichum gloeosporioides and C. acutatum in har vested apples using an antagonistic rhizobacterium Paeni-bacillus polymyxa APEC128 (APEC128). Out of 30 bacterial isolates from apple rhizosphere screened for antagonistic activity, the most effective strain was APEC128 as inferred from the size of the inhibition zone. This strain showed a greater growth in brain-heart infusion (BHI) broth compared to other growth media. There was a reduction in anthracnose symp-toms caused by the two fungal pathogens in harvested apples after their treatment with APEC128 in compari-son with non-treated control. This effect is explained by the increased production of protease and amylase by APEC128, which might have inhibited mycelial growth. In apples treated with different APEC128 sus-pensions, the disease caused by C. gloeosporioides and C. acutatum was greatly suppressed (by 83.6% and 79%, respectively) in treatments with the concen tration of 1 × 108 colony forming units (cfu)/ml com pared to other lower dosages, suggesting that the sup pression of anthracnose development on harvested apples is dose-dependent. These results indicated that APEC128 is one of the promising agents in the bio con trol of apple anthracnose, which might help to increase the shelf-life of apple fruit during the post-harvest period.

Cryopreservation of Citrus limon (L.) Burm. F Shoot Tips

이정윤,Kotnala Balaraju,백형진,윤문섭,김행훈,이영이 한국자원식물학회 2018 한국자원식물학회지 Vol.31 No.6

This study describes the successful establishment of a cryopreservation protocol for Citrus limon cultivars: ‘FrostEureka limon’ and ‘Cook Eureka limon’, using a droplet-vitrification method. The shoot tips that were excised from in vitrogrown seedlings of the two cultivars were preserved in liquid nitrogen (LN) and successfully regenerated into whole plants. Excised shoot tips were pre-cultured for 1 or 2 days in 0.3 M and 0.5 M sucrose solutions at 25℃ and incubated in a loadingsolution (LS) composed of 17.5% glycerol + 17.5% sucrose in Murashige and Skoog (MS) medium for 40 min at 25℃. Priorto direct immersion in LN for 1 h, the shoot tips were dehydrated with plant vitrification solution 2 (PVS2) at 0℃ or PVS3at 25℃. The frozen shoot tips were re-warmed and unloaded with 1.2 M sucrose in ½ MS for 30 min at 25℃. Shoot tips werepost-cultured overnight on survival medium and then micrografted onto ‘trifoliate orange’ (Poncirus trifoliate (L.) Raf. seedling rootstocks for recovery and to produce whole plants. The highest regrowth rates were 53.5% and 50.3% forcryopreserved shoot tips of ‘Frost Eureka limon’ and ‘Cook Eureka limon’, respectively, when pre-cultured in 0.3 M and 0.5M sucrose concentrations in a sequencing manner, with LS and treated with PVS2 for 60 min at 0℃. We also investigatedwhether the ammonium ion concentration on post-culture medium affected the viability of the cryopreserved Citrus shoottips. The viability of cooled samples, following culturing on woody plant media (WPM) containing ¼ ammonium nitrateovernight before micrografting, was the highest (70.3%) in ‘Frost Eureka limon’. The study described here is acost-effective and safe method to conserve Citrus fruit cultivars, for the improvement and large-scale multiplication of fruitplants and for breeding disease resistance.

A Successful Regeneration from Shoot Tips of Chrysanthemum morifolium (Ramat.) following Cryopreservation by Droplet-vitrification

이정윤,Kotnala Balaraju,백형진,윤문섭,김행훈,이영이 한국자원식물학회 2018 한국자원식물학회지 Vol.31 No.6

This study describes an efficient and widely applicable droplet-vitrification following cryopreservation for shoottips of Chrysanthemum morifolium (Ramat.) cvs. ‘Borami’ and ‘Yes morning’. The shoot tips of Chrysanthemum wereprecultured in Murashige and Skoog (MS) liquid medium supplemented with sucrose (0.3-0.7 M). Precultured explantswere treated with loading solution (LS, C6) containing glycerol 20% and sucrose 20% for 30 min and exposed todehydration solution (B5) containing 40% of glycerol and 40% of sucrose for 60 min at 25℃, and then transferred ontodroplets containing 2.5 ㎕ PVS3 on sterilized aluminum foils (4 ㎝ × 0.5 ㎝) prior to direct immersion in liquid nitrogen(LN) for 1 h. The highest regeneration rate (%) was obtained when shoot tips were precultured with treatment-2 (exposingof shoot tips to MS + 0.3M sucrose for 30 h and then treated with MS+0.5 M sucrose for 16 h) at 25℃ in both the cultivars. The viability of cooled samples, followed by culturing on NH4NO3-free MS medium for first 5 days was increased totwo-fold (80.7%) regrowth rate over those cultured on normal MS medium or MS medium containing plant growthregulators. This result shows droplet-vitrification would be a promising method for cryobanking chrysanthemumgermplasm.

Postharvest Disease Control of Colletotrichum gloeosporioides and Penicillium expansum on Stored Apples by Gamma Irradiation Combined with Fumigation

천원수,김영수,Kotnala Balaraju,김봉수,이병호,전용호 한국식물병리학회 2016 Plant Pathology Journal Vol.32 No.5

To study the control of postharvest decay caused byColletotrichum gloeosporioides and Penicillium expansum,gamma irradiation alone or in combinationwith fumigation was evaluated to extend the shelflife of apples in South Korea. An irradiation dose of2.0 kGy resulted in the maximum inhibition of C. gloeosporioides and P. expansum spore germination. The gamma irradiation dose required to reduce thespore germination by 90% was 0.22 and 0.35 kGy forC. gloeosporioides and P. expansum, respectively. Microscopicobservations revealed that when the fungalspores were treated with gamma irradiation (4.0 kGy),conidial germination was stopped completely resultingin no germ tube formation in C. gloeosporioides. Treatment with the eco-friendly fumigant ethanedinitrilehad a greater antifungal activity against C. gloeosporioidesand P. expansum in comparison with thenon-treated control under in vitro conditions. The invitro antifungal effects of the gamma irradiation andfumigation treatments allowed us to further study theeffects of the combined treatments to control postharvestdecay on stored apples. Interestingly, when appleswere treated with gamma irradiation in combinedwith fumigation, disease inhibition increased more atlower (< 0.4 kGy) than at higher doses of irradiation,suggesting that combined treatments reduced the necessaryirradiation dose in phytosanitary irradiationprocessing under storage conditions.

Biological Control of Apple Anthracnose by Paenibacillus polymyxa APEC128, an Antagonistic Rhizobacterium

Kim, Young Soo,Balaraju, Kotnala,Jeon, Yongho The Korean Society of Plant Pathology 2016 Plant Pathology Journal Vol.32 No.3

The present study investigated the suppression of the disease development of anthracnose caused by Colletotrichum gloeosporioides and C. acutatum in harvested apples using an antagonistic rhizobacterium Paenibacillus polymyxa APEC128 (APEC128). Out of 30 bacterial isolates from apple rhizosphere screened for antagonistic activity, the most effective strain was APEC128 as inferred from the size of the inhibition zone. This strain showed a greater growth in brain-heart infusion (BHI) broth compared to other growth media. There was a reduction in anthracnose symptoms caused by the two fungal pathogens in harvested apples after their treatment with APEC128 in comparison with non-treated control. This effect is explained by the increased production of protease and amylase by APEC128, which might have inhibited mycelial growth. In apples treated with different APEC128 suspensions, the disease caused by C. gloeosporioides and C. acutatum was greatly suppressed (by 83.6% and 79%, respectively) in treatments with the concentration of $1{\times}10^8$ colony forming units (cfu)/ml compared to other lower dosages, suggesting that the suppression of anthracnose development on harvested apples is dose-dependent. These results indicated that APEC128 is one of the promising agents in the biocontrol of apple anthracnose, which might help to increase the shelf-life of apple fruit during the post-harvest period.

Biological characteristics of Paenibacillus polymyxa GBR-1 involved in root rot of stored Korean ginseng

Kim, Young Soo,Kotnala, Balaraju,Kim, Young Ho,Jeon, Yongho The Korean Society of Ginseng 2016 Journal of Ginseng Research Vol.40 No.4

Background: This study aims to describe the characterization of Paenibacillus polymyxa GBR-1 (GBR-1) with respect to its positive and negative effects on plants. Methods: The morphological characteristics of GBR-1 were identified with microscopy, and subjected to Biolog analysis for identification. Bacterial population and media optimization were determined by a growth curve. The potential for GBR-1 as a growth promoting agent, to have antagonistic activity, and to have hydrolytic activity at different temperatures was assessed. The coinoculation of GBR-1 with other microorganisms and its pathogenicity on various stored plants, including ginseng, were assessed. Results: Colony morphology, endospore-bearing cells, and cell division of GBR-1 were identified by microscopy; identification was performed by utilizing the Biolog system, gas chromatography of fatty acid methyl esters (GC-FAME). GBR-1 showed the strongest antagonistic activity against fungal and bacterial pathogens. GBR-1 cell numbers were relatively higher when the cells were cultured in brain heart infusion (BHI) medium when compared with other media. Furthermore, the starch-hydrolytic activity was influenced by GBR-1 at higher temperature compared to low temperatures. GBR-1 was pathogenic to some of the storage plants. Coinoculation of GBR-1 with other pathogens causes differences in rotting on ginseng roots. A significant growth promotion was observed in tobacco seedlings treated with GBR-1 suspensions under in vitro conditions, suggesting that its volatile organic compounds (VOCs) might play a role in growth promotion. Conclusion: The results of this study indicate that GBR-1 has both positive and negative effects on ginseng root and other stored plants as a potential biocontrol agent and eliciting in vitro growth promotion.