Evaluation of Nematicidal Activity of Streptomyces yatensis KRA-28 against Meloidogyne incognita

( Eun-jae Park ),( Hyun-jae Jang ),( Chan Sun Park ),( Seung-jae Lee ),( Soyoung Lee ),( Kang-hoon Kim ),( Bong-sik Yun ),( Seung Woong Lee ),( Mun-chual Rho ) 한국미생물 · 생명공학회 2020 Journal of microbiology and biotechnology Vol.30 No.5

The root-knot nematode (Meloidogyne incognita) is an important pathogen in crop cultivation, however, few methods are available to control this parasitic roundworm. In this study, the nematicidal effects of approximately 30 Streptomyces strains isolated from soil samples of Mt. Naejang (Korea) were tested against Meloidogyne incognita, and the culture broth of the strains KRA-24 and KRA-28 exhibited approximately 75% and 85% insecticidal activity, respectively, in in vitro assays. In in vivo pot experiments, these strains reduced the number of nematodes in the soil and the number of egg masses in the roots of red peppers. The two strains also survived in the presence of insecticidal agents (0.1 to 3.0%) such as fosthiazate, ethoprophos and terbufos when they were used in parallel. The mixture of KRA-24 or KRA-28 culture broth and fosthiazate exhibited nematicidal effects that were similar to those observed when KRA-24 or KRA-28 were used alone. Our results clearly suggest that the Streptomyces strains KRA-24 and KRA-28 should be promoted as a biocontrol agent against Meloidogyne incognita.

Nematicidal activity of verrucarin A and roridin A isolated from <i>Myrothecium verrucaria</i> against <i>Meloidogyne incognita</i>

Nguyen, Loan Thi Thanh,Jang, Ja Yeong,Kim, Tae Yoon,Yu, Nan Hee,Park, Ae Ran,Lee, Seungki,Bae, Chang-Hwan,Yeo, Joo Hong,Hur, Jae-Seoun,Park, Hae Woong,Kim, Jin-Cheol Elsevier 2018 Pesticide biochemistry and physiology Vol.148 No.-

<P><B>Abstract</B></P> <P> <UL> <LI> The widespread use of synthetic nematicides has caused significant problems to the environment as well as human health. To address this issue, eco-friendly control measures, such as microbial nematicides, are being developed. During the screening of <I>Myrothecium</I> strains with nematicidal activity against the root-knot nematode (RKN) <I>Meloidogyne incognita</I>, we found that the acetone extract of <I>Myrothecium</I> sp. KACC 40321 was highly effective against hatched juveniles of <I>M. incognita</I> at 7 days after exposure. The fungus was identified as <I>Meloidogyne verrucaria</I>. Two macrocyclic trichothecenes verrucarin A and roridin A were isolated and identified as major active metabolites by bioassay-guided fractionation and instrumental analysis. When the second-stage juveniles were treated with the chemicals, no juvenile mortality was observed. However, they effectively killed juveniles from treated eggs. The hatched juvenile mortality was used to evaluate the <I>in vitro</I> nematicidal activity of the compounds against <I>M. incognita</I>. The median effective concentrations were 1.88 μg/mL for verrucarin A and 1.50 μg/mL for roridin A. Among various liquid media, commercial malt extract broth (cMEB) was found to be the best for the production of verrucarin A and roridin A, followed by potato dextrose broth. The cMEB culture filtrate effectively reduced the formation of galls and egg masses on tomato roots in a pot experiment. In addition, the culture filtrate reduced the formation of galls on the roots of melon plants and the number of RKNs in the soils under field conditions. These results suggest that <I>M. verrucaria</I> KACC 40321 can be used as a biocontrol agent against RKNs in various crops. To the best of our knowledge, this is the first study to report the effectiveness of verrucarin A and rorridin A against hatched juveniles of <I>M. incognita</I>. </LI> </UL> </P> <P><B>Highlights</B></P> <P> <UL> <LI> <I>Myrothecium verrucaria</I> KACC 40321 was highly effective against hatched juveniles of the root-knot nematode (RKN) <I>Meloidogyne incognita.</I> </LI> <LI> Verrucarin A and roridin A were isolated from <I>M. verrucaria</I> KACC 40321 as active metabolites. </LI> <LI> The fungal culture filtrate effectively reduced the formation of galls and egg masses on roots of tomato and melon plants. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Meloidogyne incognita에 살선충활성을 보이는 신규 Streptomyces netropsis의 살선충 특성 규명

장자영,최용호,주윤정,김헌,최경자,장경수,김창진,차병진,박해웅,김진철 한국식물병리학회 2015 식물병연구 Vol.21 No.2

Control of nematode has become difficult owing to the restricted use of effective soil fumigant, methyl bromide, and other non-fumigant nematicides. Therefore, it is urgently necessary to develop microbial nematicide to replace chemical nematicides. In this study, the 50% aqueous methanol extraction solution of fermentation broths of 2,700 actinomycete strains were tested for their nematicidal activity against second stage of juveniles (J2s) of Meloidogyne incognita. As the results, only the 50% aqueous methanol extraction solution of AN110065, at 20% equivalent to 10% fermentation broth, showed strong nematicidal activity with 78.9% of mortality 24 h after treatment and 94.1% of mortality at 72 h. The 16S rRNA gene sequencing showed that the strain sequence was 99.78% identical to Streptomyces netropsis. The extract of S. netropsis AN110065 fermentation broth was successively partitioned with ethyl acetate and butanol and then the ethyl acetate, butanol and water layers were investigated for their nematicidal activity against the M. incognita. At 1,000 mg/ml, ethyl acetate layer showed the strongest activity of 83.5% of juvenile mortality 72 h after treatment. The pot experiment using the fermentation broth of AN110065 on tomato plant against M. incognita displayed that it evidently suppressed gall formation at a 10-fold diluent treatment. The tomato plants treated with the fermentation broth of S. netropsis AN110065 did not show any phytotoxicity. The results suggest that S. netropsis AN110065 has a potential to serve as microbial nematicide in organic agriculture. 효과적으로 선충 방제에 사용되던 토양훈증제 methyl bromide 와 비훈중제 농약이 환경오염 문제로 인해 사용이 금지됨에 따라 선충의 방제는 어려워지고 있다. 따라서 이를 대체할 수 있는 생물 기반의 살선충제의 개발이 시급히 요구된다. 본 연구는 화학적 농약의 대안으로서 방선균을 이용한 살선충제 개발을 목적으로 2,700개의 토양방선균 배양액의 50% 메탄올 추출액의 뿌리혹선충에 대한 살선충 활성을 스크리닝하였다. AN110065 균주는 배양액 10%에 해당하는 50% 메탄올 추출액20% 처리 1일 후 78.9%의 치사 활성을 보였으며, 처리 3일 후94.1%의 살선충 활성을 보였다. 선발 균주의 분자생물학적 동정을 위해 16S rRNA sequencing 분석을 수행한 결과 S. netropsis 와 99.78%의 상동성을 보였다. S. netropsis AN110065 배양액을에틸 아세테이트, 부탄올을 이용하여 용매 분획하여 에틸 아세테이트와 부탄올, 물 추출물에 대한 살선충 활성을 조사한 결과에틸 아세테이트 추출물의 1,000 mg/ml 농도에서 83.5%로서 가장 우수한 살선충 활성을 보였다. AN110065 배양액의 토마토작물에 대한 뿌리혹선충병 방제 활성을 조사한 결과 10배 희석액 처리구에서 눈에 띄게 뿌리혹형성을 저해하였다. 이와 같은 결과로 AN110065 균주는 유기농업에서 사용할 수 있는 미생물제제로서의 가능성이 있음을 나타내었다.

Stage-Wise Identification and Analysis of miRNA from Root-Knot Nematode <i>Meloidogyne incognita</i>

Subramanian, Parthiban,Choi, In-Chan,Mani, Vimalraj,Park, Junhyung,Subramaniyam, Sathiyamoorthy,Choi, Kang-Hyun,Sim, Joon-Soo,Lee, Chang-Muk,Koo, Ja Choon,Hahn, Bum-Soo MDPI AG 2016 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.17 No.10

<P>In this study, we investigated global changes in miRNAs of <I>Meloidogyne incognita</I> throughout its life cycle. Small RNA sequencing resulted in approximately 62, 38, 38, 35, and 39 Mb reads in the egg, J2, J3, J4, and female stages, respectively. Overall, we identified 2724 known and 383 novel miRNAs (read count > 10) from all stages, of which 169 known and 13 novel miRNA were common to all the five stages. Among the stage-specific miRNAs, miR-286 was highly expressed in eggs, miR-2401 in J2, miR-8 and miR-187 in J3, miR-6736 in J4, and miR-17 in the female stages. These miRNAs are reported to be involved in embryo and neural development, muscular function, and control of apoptosis. Cluster analysis indicated the presence of 91 miRNA clusters, of which 36 clusters were novel and identified in this study. Comparison of miRNA families with other nematodes showed 17 families to be commonly absent in animal parasitic nematodes and <I>M. incognita</I>. Validation of 43 predicted common and stage-specific miRNA by quantitative PCR (qPCR) indicated their expression in the nematode. Stage-wise exploration of <I>M. incognita</I> miRNAs has not been carried out before and this work presents information on common and stage-specific miRNAs of the root-knot nematode.</P>

Differential Metabolic Profiles during the Developmental Stages of Plant-Parasitic Nematode <i>Meloidogyne incognita</i>

Subramanian, Parthiban,Oh, Byung-Ju,Mani, Vimalraj,Lee, Jae Kook,Lee, Chang-Muk,Sim, Joon-Soo,Koo, Ja Choon,Hahn, Bum-Soo MDPI AG 2017 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.18 No.7

<P><I>Meloidogyne incognita</I> is a common root-knot nematode with a wide range of plant hosts. We aimed to study the metabolites produced at each stage of the nematode life cycle to understand its development. Metabolites of <I>Meloidogyne incognita</I> were extracted at egg, J2, J3, J4, and female stages and 110 metabolites with available standards were quantified using CE-TOF/MS. Analyses indicated abundance of stage-specific metabolites with the exception of J3 and J4 stages which shared similar metabolic profiles. The egg stage showed increased abundance in glycolysis and energy metabolism related metabolites while the J2 metabolites are associated with tissue formation, motility, and neurotransmission. The J3 and J4 stages indicated amino acid metabolism and urea cycle- related metabolites. The female stage was characterized with polyamine synthesis, antioxidant activity, and synthesis of reproduction related metabolites. Such metabolic profiling helps us understand the dynamic physiological changes related to each developmental stage of the root-knot nematode life cycle.</P>

Nematicidal Activities of 4-Quinolone Alkaloids Isolated from the Aerial Part of <i>Triumfetta grandidens</i> against <i>Meloidogyne incognita</i>

Jang, Ja Yeong,Le Dang, Quang,Choi, Yong Ho,Choi, Gyung Ja,Jang, Kyoung Soo,Cha, Byeongjin,Luu, Ngoc Hoang,Kim, Jin-Cheol American Chemical Society 2015 Journal of agricultural and food chemistry Vol.63 No.1

<P>The methanol extract of the aerial part of <I>Triumfetta grandidens</I> (Tiliaceae) was highly active against <I>Meloidogyne incognita</I>, with second-stage juveniles (J2s) mortality of 100% at 500 μg/mL at 48 h post-exposure. Two 4-quinolone alkaloids, waltherione E (<B>1</B>), a new alkaloid, and waltherione A (<B>2</B>), were isolated and identified as nematicidal compounds through bioassay-guided fractionation and instrumental analysis. The nematicidal activities of the isolated compounds against <I>M. incognita</I> were evaluated on the basis of mortality and effect on egg hatching. Compounds <B>1</B> and <B>2</B> exhibited high mortalities against J2s of <I>M. incognita</I>, with EC<SUB>50</SUB> values of 0.09 and 0.27 μg/mL at 48 h, respectively. Compounds <B>1</B> and <B>2</B> 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 <I>T. grandidens</I> showed that it completely suppressed the formation of gall on roots of plants at a concentration of 1000 μg/mL. These results suggest that <I>T. grandidens</I> and its bioactive 4-quinolone alkaloids can be used as a potent botanical nematicide in organic agriculture.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jafcau/2015/jafcau.2015.63.issue-1/jf504572h/production/images/medium/jf-2014-04572h_0005.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/jf504572h'>ACS Electronic Supporting Info</A></P>

Nematicidal Activity of Kojic Acid Produced by Aspergillus oryzae against Meloidogyne incognita

( Tae Yoon Kim ),( Ja Yeong Jang ),( Sun Jeong Jeon ),( Hye Won Lee ),( Chang-hwan Bae ),( Joo Hong Yeo ),( Hyang Burm Lee ),( In Seon Kim ),( Hae Woong Park ),( Jin-cheol Kim ) 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.8

The fungal strain EML-DML3PNa1 isolated from leaf of white dogwood (Cornus alba L.) showed strong nematicidal activity with juvenile mortality of 87.6% at a concentration of 20% fermentation broth filtrate at 3 days after treatment. The active fungal strain was identified as Aspergillus oryzae, which belongs to section Flavi, based on the morphological characteristics and sequence analysis of the ITS rDNA, calmodulin (CaM), and β-tubulin (BenA) genes. The strain reduced the pH value to 5.62 after 7 days of incubation. Organic acid analysis revealed the presence of citric acid (515.0 mg/kg), malic acid (506.6 mg/kg), and fumaric acid (21.7 mg/kg). The three organic acids showed moderate nematicidal activities, but the mixture of citric acid, malic acid, and fumaric acid did not exhibit the full nematicidal activity of the culture filtrate of EML- DML3PNa1. Bioassay-guided fractionation coupled with 1H- and 13C-NMR and EI-MS analyses led to identification of kojic acid as the major nematicidal metabolite. Kojic acid exhibited dose-dependent mortality and inhibited the hatchability of M. incognita, showing EC50 values of 195.2 μg/ml and 238.3 μg/ml, respectively, at 72 h postexposure. These results suggest that A. oryzae EML-DML3PNa1 and kojic acid have potential as a biological control agent against M. incognita.

Meloidogyne incognita에 대한 Streptomyces flavogriseus KRA15-528의 살선충활성

오미라,한재우,최정섭,최용호,장경수,최경자,김헌 한국식물병리학회 2016 식물병연구 Vol.22 No.4

Plant disease caused by root-knot nematode is a major problem in crop production. Using of chemical pesticides, one of the most efficient methods to control nematodes, have raised issues in toxicity to humans and animals and environmental pollution. In this study, to select actinomycete strains that have potential to serve as a microbial agent for control of nematodes, we investigated nematicidal activity of culture broth from 670 Streptomyces isolates. A culture filtrate of KRA15-528 isolate that was identified as S. flavogriseus on the basis of 16S rRNA sequence analysis, showed strong nematicidal activity against second stage of juveniles of Meloidogyne incognita and inhibited egg hatching; exposure to 10% of culture filtrate resulted in 71% juvenile mortality at 48 hours afters treatment and suppressed egg hatching by 54% at 9 days after treatment. When the KRA15-528 culture filtrate was partitioned with ethyl acetate and butanol, ethyl acetate layer exclusively showed strong activity; 91%, 53%, 30% of mortality at 1,000, 500, 250 μg/ml, respectively. Additionally, the culture filtrate suppressed gall formation on cucumber plant by M. incognita with no phytotoxicity. These results suggest that S. flavogriseus KRA15-528 has potential to serve as a microbial nematicide for the control of root-knot nematode disease.

Evaluation of oil dispersion formulation of nematophagus fungus, Pochonia chlamydosporia against root-knot nematode, Meloidogyne incognita in cucumber

Swarnakumari N.,Sindhu R.,Thiribhuvanamala G.,Rajaswaminathan V. 한국응용곤충학회 2020 Journal of Asia-Pacific Entomology Vol.23 No.4

Root-knot nematode, Meloidogyne incognita is considered as one of the major non-insect pests of crops. The management of these root feeders becomes highly challenging due to a strong host-parasitic relationship. Pochonia chlamydosporia is a nematophagus fungus that colonizes eggs of nematodes. This study aimed to test the efficacy of P. chlamydosporia (NAIMCC-SF0039) against M. incognita. An oil dispersion formulation of P. chlamydosporia was prepared using emulsifiers and vegetable oil. This formulation had a shelf-life of 90 days (3.3 × 10 8 CFU/mL) at room temperature (28 ± 1 °C). The inhibitory effect of oil formulation was tested against M. incognita by inoculating it on the egg mass. We found that colonization of the gelatinous matrix occurred on the third day of inoculation followed by complete egg parasitization on the seventh day. A greenhouse trial was laid out to evaluate the biocontrol potential of P. chlamydosporia in cucumber (Cucumis sativus). The results showed that the application of talc formulation of P. chlamydosporia at the rate of 1 kg per acre during planting, followed by delivery of 1 L of oil dispersion formulation through drip lines at 30-day intervals caused the highest reduction of nematode infestation. This treatment recorded 67.9 and 57.5% reduction in egg masses and soil nematode population respectively than that of control.

토마토 뿌리혹선충 Meloidogyne incognita에 치사력이 있는 Bacillus thuuingiensis Bt TH109 균주의 분리 및 특성

이광배,김광현,김영희 한국미생물 · 생명공학회 1994 한국미생물·생명공학회지 Vol.22 No.3

In order to microbially control root-knot nematode(Meloidogyne incognita) in tomato, a strain BtTH109 of Bacillus thuringiensis producing root-knot nematocidal toxin was isolated. The strain BtTH109 was identified B. thuringiensis subsp. indiana(serotype 16) based on flagella antigenicity, biochemical properties, and morphological charcateristics. The strain BtTH109 have extracellularly produced a root-knot nematocidal toxin, which was very toxic against not only egghatch but also the 2nd-nematode larva of root-knot nematode in vitro.