Development of Real-time PCR Assay Based on Hydrolysis Probe for Detection of Epichloë spp. and Toxic Alkaloid Synthesis Genes

이기원,우제훈,송요욱,Md Atikur Rahman,이상훈 한국초지조사료학회 2022 한국초지조사료학회지 Vol.42 No.3

Fescues, which are widely cultivated as grasses and forages around the world, are often naturally infected with the endophyte, Epichloë. This fungus, transmitted through seeds, imparts resistance to drying and herbivorous insects in its host without causing any external damage, thereby contributing to the adaptation of the host to the environment and maintaining a symbiosis. However, some endophytes, such as E. coenophialum synthesize ergovaline or lolitrem B, which accumulate in the plant and impart anti-mammalian properties. For example, when livestock consume excessive amounts of grass containing toxic endophytes, problems associated with neuromuscular abnormalities, such as convulsions, paralysis, high fever, decreased milk production, reproductive disorders, and even death, can occur. Therefore, pre-inoculation with non-toxic endogenous fungi or management with endophyte-free grass is important in preventing damage to livestock and producing high-quality forage. To date, the diagnosis of endophytes has been mainly performed by observation under a microscope following staining, or by performing an immune blot assay using a monoclonal antibody. Recently, the polymerase chain reaction (PCR)-based molecular diagnostic method is gaining importance in the fields of agriculture, livestock, and healthcare given the method’s advantages. These include faster results, with greater accuracy and sensitivity than those obtained using conventional diagnostic methods. For the diagnosis of endophytes, the nested PCR method is the only available option developed; however, it is limited by the fact that the level of toxic alkaloid synthesis cannot be estimated. Therefore, in this study, we aimed to develop a triplex real-time PCR diagnostic method that can determine the presence or absence of endophyte infection using DNA extracted from seeds within 1 h, while simultaneously detecting easD and LtmC genes, which are related to toxic alkaloid synthesis. This new method was then also applied to real field samples.

Identification and characterization of type III polyketide synthase genes from culturable endophytes of ethnomedicinal plants

Manoharan, Gomathi,Sairam, Thiagarajan,Thangamani, Rajesh,Ramakrishnan, Dhivya,K.Tiwari, Manish,Lee, Jung-Kul,Marimuthu, Jeya IPC Science and Technology Press 2019 Enzyme and microbial technology Vol.131 No.-

<P><B>Abstract</B></P> <P>Endophytic fungi provide benefits to host plants by producing a diverse class of secondary metabolites (natural products). Arrays of polyketide natural products are synthesized by specific classes of polyketide synthases (PKS I, II and III) in host organisms. In the present study, we attempt to screen and identify type III PKSs in culturable fungal endophytes isolated from the ethno medicinal plants including <I>Arbus precatorius</I>, <I>Bacopa monnieri,Citrus aurantifolia</I> and <I>Datura metel</I> to detect the genetic potential of endophytic fungi in producing bioactive compounds. A total of seventeen endophytic fungal strains belonging to eight genera were identified using fungal morphology and rDNA-ITS phylogenetic analyses. A CODEHOP-PCR based strategy was followed to design degenerate primers for the screening of type III PKS genes from fungal endophytes. We had successfully amplified partial PKS genes from eight endophytes. The amplified PKS sequences showed 60–99% identity to already characterized/putative PKS genes. From the partial sequence of FiPKS from <I>Fusarium incarnatum</I> BMER1, a full-length gene was amplified, cloned and characterized. FiPKScDNA was cloned and expressed in <I>E. coli</I> Lemo21 (DE3) and the purified protein was shown to produce pyrones and resorcinols using acyl-CoA thioesters as substrates. FiPKS showed the highest catalytic efficiency of 7.6 × 10<SUP>4</SUP> s<SUP>−1</SUP> M<SUP>-1</SUP> with stearoyl CoA as a starter unit. This study reports the identification and characterization of type III PKS from endophytes of medicinal plants by CODEHOP PCR.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A CODEHOP PCR based screening method was employed for type III polyketide synthase gene identification in fungal endophytes. </LI> <LI> By this approach, partial type III PKS genes from eight fungal endophytes were amplified and sequenced. </LI> <LI> FiPKS gene from Fusarium incarnatum BMER1, an endophyte of Bacopa monnieri was cloned and functionally characterized. </LI> <LI> FiPKS produced pyrones and resorcinols with the highest catalytic efficiency of 7.6 x 104 s-1 M-1towards stearoyl CoA. </LI> </UL> </P>

Comparison of Endophytic Microbial Community in Kiwifruit Plant Cultivars

조경준,김민정,권영호,곽연식 한국식물병리학회 2018 Plant Pathology Journal Vol.34 No.4

The microbiome makes a significant contribution to plant health and endophytes may generate positive effects for the host. However, there is a limited knowledge available concerning the kiwifruit endophyte. Therefore, we discuss endophyte microbiome community structures among the kiwifruit cultivars. Total reads numbered 17620 in cv. Hayward, 11515 in cv. Haegeum and 13613 in cv. Jecygold. The number of OTUs was follows: 112 in cv. Hageum; and 87 in cvs. Hayward and Jecygold. Most of the identified OTUs were phylum Proteobacteria and it emerged that Actinobacteria, Firmicute and Bacteroidetes were mainly present. While the largest difference in Proteobacteria content is between cv. Haegeum and cv. Jecygold, they shared more OTUs than cv. Hayward. As well, this study revealed the presence of remarkably dominated OTU of Pseudomonas spp. in kiwifruit sap regardless of cultivars. To the best of our knowledge, this study is the first tone to investigate the kiwifruit endophyte-microbiome community.

Comparison of Endophytic Microbial Community in Kiwifruit Plant Cultivars

Cho, Gyeongjun,Kim, Min-jung,Kwon, Youngho,Kwak, Youn-Sig The Korean Society of Plant Pathology 2018 Plant Pathology Journal Vol.34 No.4

The microbiome makes a significant contribution to plant health and endophytes may generate positive effects for the host. However, there is a limited knowledge available concerning the kiwifruit endophyte. Therefore, we discuss endophyte microbiome community structures among the kiwifruit cultivars. Total reads numbered 17620 in cv. Hayward, 11515 in cv. Haegeum and 13613 in cv. Jecygold. The number of OTUs was follows: 112 in cv. Hageum; and 87 in cvs. Hayward and Jecygold. Most of the identified OTUs were phylum Proteobacteria and it emerged that Actinobacteria, Firmicute and Bacteroidetes were mainly present. While the largest difference in Proteobacteria content is between cv. Haegeum and cv. Jecygold, they shared more OTUs than cv. Hayward. As well, this study revealed the presence of remarkably dominated OTU of Pseudomonas spp. in kiwifruit sap regardless of cultivars. To the best of our knowledge, this study is the first tone to investigate the kiwifruit endophyte-microbiome community.

Fungal Endophytes of Alnus incana ssp. rugosa and Alnus alnobetula ssp. crispa and Their Potential to Tolerate Heavy Metals and to Promote Plant Growth

( Steve Lalancette ),( Sylvain Lerat ),( S_ebastien Roy ),( Carole Beaulieu ) 한국균학회 2019 Mycobiology Vol.47 No.4

Soil contamination by metals is of particular interest, given that their retention times within the profile can be indefinite. Thus, phytostabilization can be viewed as a means of limiting metal toxicity in soils. Due to their ability to grow on contaminated soils, alders have repeatedly been used as key species in phytostabilization efforts. Alder ability to grow on contaminated sites stems, in part, from its association with microbial endophytes. This work emphasizes the fungal endophytes populations associated with Alnus incana ssp. rugosa and Alnus alnobetula ssp. crispa (previously A. viridis ssp. crispa) under a phytostabilization angle. Fungal endophytes were isolated from alder trees that were growing on or near disturbed environments; their tolerances to Cu, Ni, Zn, and As, and acidic pH (4.3, 3, and 2) were subsequently assessed. Cryptosporiopsis spp. and Rhizoscyphus spp. were identified as fungal endophytes of Alnus for the first time. When used as inoculants for alder, some isolates promoted plant growth, while others apparently presented antagonistic relationships with the host plant. This study reports the first step in finding the right fungal endophytic partners for two species of alder used in phytostabilization of metal-contaminated mining sites.

Four Endophytic Ascomycetes New to Korea: Cladosporium anthropophilum, C. pseudocladosporioides, Daldinia eschscholtzii, and Nigrospora chinensis

( Dong Jae Lee ),( Jae Sung Lee ),( Hyang Burm Lee ),( Young-joon Choi ) 한국균학회 2019 韓國菌學會誌 Vol.47 No.3

Ascomycota is the largest phylum of the Fungi, including approximately 6,600 genera. They are often isolated from soils, indoor air, and freshwater environments, but also from plants as pathogens or endophytes. In this study, four species of Ascomycota (two of Cladosporium and one of each Daldinia and Nigrospora) were collected from the leaves of four woody plants (Camellia japonica, Ginkgo biloba, Quercus sp., Vitis vinifera). Their cultural characteristics were investigated on five different media (PDA, V8A, CMA, MEA, CZA) at 3 days after incubation at 25°C in darkness. BLASTn search and phylogenetic analysis were performed using the internal transcribed spacer (ITS) rDNA sequences, in addition to tef1 gene sequences for Cladosporium species. Based on the cultural, morphological, and phylogenetic data, the isolates were identified as Cladosporium anthropophilum, Cladosporium pseudocladosporioides, Daldinia eschscholtzii, and Nigrospora chinensis. Previously, some members of Cladosporium and Nigrospora have been recorded as endophytes inhabiting the leaves and stems of various plants, whereas Daldinia eschscholtzii is a wood-inhabiting endophyte or wood-decaying fungus. To our knowledge, this is the first report of these four ascomycetes in Korea.

Highly diverse endophytes in roots of Cycas bifida (Cycadaceae), an ancient but endangered gymnosperm

Ying Zheng,Tzen-Yuh Chiang,Chao-Li Huang,Xun Gong 한국미생물학회 2018 The journal of microbiology Vol.56 No.5

As an ancient seed plant, cycads are one of the few gymnosperms that develop a root symbiosis with cyanobacteria, which has allowed cycads to cope with harsh geologic and climatic conditions during the evolutionary process. However, the endophytic microbes in cycad roots remain poorly identified. In this study, using next-generation sequencing techniques, we investigated the microbial diversity and composition of both the coralloid and regular roots of Cycas bifida (Dyer) K.D. Hill. Highly diverse endophytic communities were observed in both the coralloid and regular roots. Of the associated bacteria, the top five families were the Nostocaceae, Sinobacteraceae, Bradyrhizobiaceae, Bacillaceae, and Hyphomicrobiaceae. The Nectriaceae, Trichocomaceae, and Incertae sedis were the predominant fungal families in all root samples. A significant difference in the endophytic bacterial community was detected between coralloid roots and regular roots, but no difference was observed between the fungal communities in the two root types. Cyanobacteria were more dominant in coralloid roots than in regular roots. The divergence of cycad root structures and the modified physiological processes may have contributed to the abundance of cyanobionts in coralloid roots. Consequently, the colonization of cyanobacteria inhibits the assemblage of other endophytes. Our results contribute to an understanding of the species diversity and composition of the cycad-endophyte microbiome and provide an abbreviated list of potential ecological roles of the core microbes present.

Defense response of resistant host Impatiens balsamina to the parasitic angiosperm Cuscuta japonica

이규배,Judith A. Jernstedt 한국식물학회 2013 Journal of Plant Biology Vol.56 No.3

The response of the stem of a resistant host (Impatiens baslamina) to infection by the parasitic flowering plant Cuscuta japonica was studied with light and electron microscopy. The intra- and interfascicular cambial cells in the host stem first reacted to the penetrating upper haustorium by dividing, and the differentiation of the host xylem (vascular) tissues proceeded toward interfascicular areas from vascular bundles. When the host vascular tissue was invaded by the endophyte (haustorial portion in the host stem), the host xylem was displaced, and host vessels became occluded with parenchyma cells, resulting in tyloses. As the parasitism progressed, areas of the host stem penetrated by the endophyte became swollen via secondary growth and cell division in the parenchymatous cortex, pith, and interfascicular areas. During this intrusion by the endophyte, darkly stained necrotic reactions were detected at the interface between the host tissue and the invading endophyte. The results suggested that in the host tissues penetrated by the parasite, the formation of secondary tissue and swellings caused by active cell division of ground tissue and host vessel occlusion by tyloses constitute the host structural defense against the parasite.

Endophyte-assisted phytoremediation of Organic pollutants

Jun Won Kang,Yeong Dae Park 경상대학교 농업생명과학연구원 2019 농업생명과학연구 Vol.53 No.5

Rapid industrialization and urbanization have generated huge amount of environmental pollution. Especially, synthetic organic chemicals have been a serious international problem for over half a century due to their toxic and hazardous chemicals. Eco-friendly strategies for removing the chemicals from the soil and water are becoming a top priority around the world and biological treatment such as phytoremediation and bioremediation is less expensive and more sustainable than other conventional remediation techniques. Recently, many pollutant diminishing microbial endophytes have been discovered from various plants grown in contaminated area and the function of microbes to improve phytoremediation of organic pollutants has been reported. Thus, we classify synthetic organic pollutants into groups of similar compounds and discuss the contribution of endophytes to enhance phytoremediation.

Endophyte-assisted phytoremediation of Organic pollutants

강준원,박영대 경상국립대학교 농업생명과학연구원 2019 농업생명과학연구 Vol.53 No.5

Rapid industrialization and urbanization have generated huge amount of environmental pollution. Especially, synthetic organic chemicals have been a serious international problem for over half a century due to their toxic and hazardous chemicals. Eco-friendly strategies for removing the chemicals from the soil and water are becoming a top priority around the world and biological treatment such as phytoremediation and bioremediation is less expensive and more sustainable than other conventional remediation techniques. Recently, many pollutant diminishing microbial endophytes have been discovered from various plants grown in contaminated area and the function of microbes to improve phytoremediation of organic pollutants has been reported. Thus, we classify synthetic organic pollutants into groups of similar compounds and discuss the contribution of endophytes to enhance phytoremediation.