Isolation and characterization of Paenibacillus peoriae JC-3jx from Dendrobium nobile.

Dendrobium is a rich source of high-value natural components. Endophytic fungi are well studied, yet bacteria research is limited. In this study, endophytic bacteria from Dendrobium nobile were isolated using an improved method, showing inhibition of pathogens and growth promotion. JC-3jx, identified as Paenibacillus peoriae, exhibited significant inhibitory activity against tested fungi and bacteria, including Escherichia coli. JC-3jx also promoted corn seed rooting and Dendrobium growth, highlighting its excellent biocontrol and growth-promoting potential.

Antimicrobial activity and comparative metabolomic analysis of Priestia megaterium strains derived from potato and dendrobium.

The growth of endophytic bacteria is influenced by the host plants and their secondary metabolites and activities. In this study, P. megaterium P-NA14 and P. megaterium D-HT207 were isolated from potato tuber and dendrobium stem respectively. They were both identified as Priestia megaterium. The antimicrobial activities and metabolites of both strains were explored. For antimicrobial activities, results showed that P. megaterium P-NA14 exhibited a stronger inhibition effect on the pathogen of dendrobium, while P. megaterium D-HT207 exhibited a stronger inhibition effect on the pathogen of potato. The supernatant of P. megaterium P-NA14 showed an inhibition effect only on Staphylococcus aureus, while the sediment of P. megaterium D-HT207 showed an inhibition effect only on Escherichia coli. For metabolomic analysis, the content of L-phenylalanine in P. megaterium P-NA14 was higher than that of P. megaterium D-HT207, and several key downstream metabolites of L-phenylalanine were associated with inhibition of S. aureus including tyrosine, capsaicin, etc. Therefore, we speculated that the different antimicrobial activities between P. megaterium P-NA14 and P. megaterium D-HT207 were possibly related to the content of L-phenylalanine and its metabolites. This study preliminarily explored why the same strains isolated from different hosts exhibit different activities from the perspective of metabolomics.

Complete genome of Sphingomonas paucimobilis ZJSH1, an endophytic bacterium from Dendrobium officinale with stress resistance and growth promotion potential.

Sphingomonas paucimobilis ZJSH1 is an endophytic bacterium isolated from the roots of Dendrobium officinale with the ability to promote plant growth. It was found that the genome of strain ZJSH1 had gene fragment rearrangement compared with the genomes of the other four strains of S. paucimobilis, and the genome was integrated with phage genes. Functional analysis showed that the strain contained colonization-related genes, chemotaxis and invasion. A variety of genes encoding active materials, such as hormones (IAA, SA, ABA and zeaxanthin), phosphate cycle, antioxidant enzymes, and polysaccharides were identified which provide the strain with growth promotion and stress-resistant characteristics. Experiments proved that S. paucimobilis ZJSH1 grew well in media containing 80 g/L sodium chloride, 240 g/L polyethylene glycol and 800 µmol/L Cd2+, indicating its potential for resistance to stresses of salt, drought and cadmium, respectively. S. paucimobilis ZJSH1 is the only endophytic bacterium of this species that has been reported to promote plant growth. The analysis of its genome is conducive to understanding its growth-promoting mechanism and laying a foundation for the development and utilization of this species in the field of agriculture.

Improved Cultivation and Isolation of Diverse Endophytic Bacteria Inhabiting Dendrobium Roots by Using Simply Modified Agar Media.

Dendrobium plants are members of the family Orchidaceae, many of which are endangered orchids with ornamental and medicinal values. Dendrobium endophytic microbes have attracted attention for the development of strategies for plant protection and utilization of medicinal principles. However, the role of endophytic bacteria is poorly elucidated due to the lack of their successful cultivation. This study obtained a total of 749 endophytic isolates from Dendrobium roots using solid media prepared by simply modified methods (separate sterilization of phosphate and agar [PS] and use of gellan gum as a gelling reagent [GG]) and by a conventional method of autoclaving the phosphate and agar together (PT method). Notably, based on a comparison of 16S rRNA gene sequences between the isolates and the Dendrobium root endophyte community, we successfully retrieved more than 50% (17 out of 30) of the predominant endophytic bacterial operational taxonomic units (OTUs) using PS and GG media, which is a much higher recovery rate than that of PT medium (16.7%). We further found that a number of recalcitrant bacteria, including phylogenetically novel isolates and members of even the rarely cultivated phyla Acidobacteriota and Verrucomicrobiota, were obtained only when using PS and/or GG medium. Intriguingly, the majority of these recalcitrant bacteria formed colonies faster on PS or GG medium than on PT medium, which may have contributed to their successful isolation. Taken together, this study succeeded in isolating a wide variety of Dendrobium endophytic bacteria, including predominant ones using PS and GG media, and enables performance of future studies to clarify their unknown roles associated with the growth of Dendrobium plants. IMPORTANCE Dendrobium endophytic bacteria are of great interest since their functions may contribute to the protection of endangered orchids with ornamental and medicinal values. To understand and reveal the "true roles" of the endophytes, obtaining those axenic cultures is necessary even in the metagenomic era. However, no effective methods for isolating a variety of endophytic bacteria have been established. This study first demonstrated that the use of simply modified medium is quite effective and indeed allows the isolation of more than half of the predominant endophytic bacteria inhabiting Dendrobium roots. Besides, even phylogenetically novel and/or recalcitrant endophytic bacteria were successfully obtained by the same strategy. The obtained endophytic bacteria could serve as "living material" for elucidating their unprecedented functions related to the conservation of endangered orchid plants. Furthermore, the culture method used in this study may enable the isolation of various endophytic bacteria dominating not only in orchid plants but also in other useful plants.

Isolation and identification of endophytic fungi from Dendrobium huoshanense with their antibacterial and anti-inflammatory activities.

To examine the symbiotic relationship between Dendrobium huoshanense and its endophytic fungi, a total of 168 endophytic fungi were isolated and classified into 18 genera and 36 species via using pure culture and techniques of molecular biology. Among them, Stagonosporopsis oculihominis, Alternaria eichhorniae, Phyllosticta aristolochiicola, Aspergillus flavus and Fusarium lactis were the dominant genera. Further, the secondary metabolites produced by the dominant genera were screened for antibacterial and anti-inflammatory activities. The secondary metabolites of Stagonosporopsis oculihominis showed good inhibitory activity against Staphylococcus aureus and the release of nitric oxide (NO). Three benzene ring derivatives were also identified using chromatographic and spectroscopic techniques, including (Z)-1- hydroxy-4-(2-nitroethenyl)-benzene (1), p-hydroxybenzaldehyde (2) and p-hydroxyphenylacetic acid (3). These findings indicated that the endophytic fungi of Dendrobium huoshanense are diverse and their dominant genera exert similar anti-inflammatory and antibacterial effects as those of the host plant, which provided a scientific basis for the subsequent investigation of the medicinal value of Dendrobium huoshanense.

Potential Specificity Between Mycorrhizal Fungi Isolated from Widespread Dendrobium spp. and Rare D. huoshanense Seeds.

In nature, orchid seed germination and seedling development depend on compatible mycorrhizal fungi. Mycorrhizal generalist and specificity affect the orchid distribution and rarity. Here, we investigated the specificity toward fungi in the rare D. huoshanense by mycorrhizal fungal isolation and symbiotic germination in vitro. Twenty mycorrhizal fungal strains were isolated from the roots of adult Dendrobium spp. (six and 12 strains from rare D. huoshanense and widespread D. officinale, respectively, and two strains from D. nobile and D. moniliforme, respectively) and 13 strains belong to Tulasnellaceae and seven strains belong to Serendipitaceae. Germination trials in vitro revealed that all 20 tested fungal strains can stimulate seed germination of D. huoshanense, but only nine strains (~ 50%) can support it up to the seedling stage. This finding indicates that generalistic fungi are important for early germination, but only a few can maintain a symbiosis with host in seedling stage. Thus, a shift of the microbial community from seedling to mature stage probably narrows the D. huoshanense distribution range. In addition, to further understand the relationship between the fungal capability to promote seed germination and fungal enzyme activity, we screened the laccase and pectase activity. The results showed that the two enzymes activities of fungi cannot be directly correlated with their germination-promoting activities. Understanding the host specificity degree toward fungi can help to better interpret the limited geographic distribution of D. huoshanense and provides opportunities for in situ and ex situ conservation and reintroduction programs.

What role does the seed coat play during symbiotic seed germination in orchids: an experimental approach with Dendrobium officinale.

BACKGROUND: Orchids require specific mycorrhizal associations for seed germination. During symbiotic germination, the seed coat is the first point of fungal attachment, and whether the seed coat plays a role in the identification of compatible and incompatible fungi is unclear. Here, we compared the effects of compatible and incompatible fungi on seed germination, protocorm formation, seedling development, and colonization patterns in Dendrobium officinale; additionally, two experimental approaches, seeds pretreated with NaClO to change the permeability of the seed coat and fungi incubated with in vitro-produced protocorms, were used to assess the role of seed coat played during symbiotic seed germination. RESULTS: The two compatible fungi, Tulasnella sp. TPYD-2 and Serendipita indica PI could quickly promote D. officinale seed germination to the seedling stage. Sixty-two days after incubation, 67.8 ± 5.23% of seeds developed into seedlings with two leaves in the PI treatment, which was significantly higher than that in the TPYD-2 treatment (37.1 ± 3.55%), and massive pelotons formed inside the basal cells of the protocorm or seedlings in both compatible fungi treatments. In contrast, the incompatible fungus Tulasnella sp. FDd1 did not promote seed germination up to seedlings at 62 days after incubation, and only a few pelotons were occasionally observed inside the protocorms. NaClO seed pretreatment improved seed germination under all three fungal treatments but did not improve seed colonization or promote seedling formation by incompatible fungi. Without the seed coat barrier, the colonization of in vitro-produced protocorms by TPYD-2 and PI was slowed, postponing protocorm development and seedling formation compared to those in intact seeds incubated with the same fungi. Moreover, the incompatible fungus FDd1 was still unable to colonize in vitro-produced protocorms and promote seedling formation. CONCLUSIONS: Compatible fungi could quickly promote seed germination up to the seedling stage accompanied by hyphal colonization of seeds and formation of many pelotons inside cells, while incompatible fungi could not continuously colonize seeds and form enough protocorms to support D. officinale seedling development. The improvement of seed germination by seed pretreatment may result from improving the seed coat hydrophilicity and permeability, but seed pretreatment cannot change the compatibility of a fungus with an orchid. Without a seed coat, the incompatible fungus FDd1 still cannot colonize in vitro-produced protocorms or support seedling development. These results suggest that seed coats are not involved in symbiotic germination in D. officinale.

Tiankengomelania guangxiense, gen. et sp. nov., a dark septate endophytic fungus, promotes the growth of the medicinal orchid Dendrobium officinale.

Dendrobium officinale (Orchidaceae) is a traditional Chinese medicinal plant. Its growth is slow, however many dark septate endophytic fungi (DSEs) are considered useful to plant growth and as biocontrol agents against plant pathogens. The goals of this study were to identify a new DSE and evaluate its plant-growth promotion characteristics. Based on morphological and molecular phylogenetic evidence, a DSE fungal strain TK815 isolated from Dashiwei Tiankengs in Leye county Guangxi Province, China, was classified as a novel genus in the order Cheatothyriales, namely Tiankengomelania gen. nov. typified with T. guangxiense sp. nov. Tiankengomelania guangxiense TK815 can significantly promote the growth of D. officinale in stem length (11.25%), seedling height (16.97%), root length (10.34%), and dry weight (41.05%). This study discovered, described, and illustrated a new DSE fungus, and evaluated its biological function in contributing to the growth and production of the Chinese medicinal plant D. officinale.

Taxonomic and functional diversity of Dendrobium officinale microbiome in Danxia habitat.

AIMS: Microbial communities that inhabit plants are crucial for plant survival and well-being including growth in stressful environments. The medicinal plant, Dendrobium officinale grows in the barren soils of the Danxia Habitat. However, the microbiome composition and functional potential for growth of this plant in this environment are still unexplored. METHODS AND RESULTS: In this study, we analysed the taxonomic and functional diversity of the D. officinale Microbiome by metagenomic sequencing of both rhizosphere and endosphere samples. A total of 155 phyla, 122 classes, 271 orders, 620 families and 2194 genera were identified from all samples. The rhizospheric microbes (DXRh) were mainly composed of Proteobacteria and Acidobacteria, while Basidiomycota and Ascomycota were the most dominant phyla in root endosphere (DXRo) and stem endosphere (DXS), respectively. Most of the dominant microbial communities had been reported to have diverse functional potentials that can help plant growth and development in stressful and nutrient-deprived ecological environmental. These include plant growth promoting rhizobacteria (PGPR) such as Massilia, Pseudomonas, Bradyrhizobium, Klebsiella, Streptomyces, Leclercia, Paenibacillus, Frankia and Enterobacter in the DXRh, Tulasnella and Serendipita in the DXRo, Colletotrichum and Burkholderia in the DXS and Paraburkholderia, Rhizophagus and Acetobacter in endosphere. Analysis using the KEGG, eggNOG and CAZy databases showed that metabolic pathways such as carbohydrate metabolism, amino acid metabolism, energy metabolism, genetic information processing and environmental information processing are significantly abundant, which may be related to the survival, growth and development of D. officinale in a stressful environment. CONCLUSIONS: We speculated that the microbial community with diverse taxonomic structures and metabolic functions inhabiting in different niches of plants supports the survival and growth of D. officinale in the stressful environment of Danxia Habitat. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided an important data resource for microbes associated with D. officinale and theoretical foundation for further studies.

JA signal-mediated immunity of Dendrobium catenatum to necrotrophic Southern Blight pathogen.

BACKGROUND: Dendrobium catenatum belongs to the Orchidaceae, and is a precious Chinese herbal medicine. In the past 20 years, D. catenatum industry has developed from an endangered medicinal plant to multi-billion dollar grade industry. The necrotrophic pathogen Sclerotium delphinii has a devastating effection on over 500 plant species, especially resulting in widespread infection and severe yield loss in the process of large-scale cultivation of D. catenatum. It has been widely reported that Jasmonate (JA) is involved in plant immunity to pathogens, but the mechanisms of JA-induced plant resistance to S. delphinii are unclear. RESULTS: In the present study, the role of JA in enhancing D. catenatum resistance to S. delphinii was investigated. We identified 2 COI1, 13 JAZ, and 12 MYC proteins in D. catenatum genome. Subsequently, systematic analyses containing phylogenetic relationship, gene structure, protein domain, and motif architecture of core JA pathway proteins were conducted in D. catenatum and the newly characterized homologs from its closely related orchid species Phalaenopsis equestris and Apostasia shenzhenica, along with the well-investigated homologs from Arabidopsis thaliana and Oryza sativa. Public RNA-seq data were investigated to analyze the expression patterns of D. catenatum core JA pathway genes in various tissues and organs. Transcriptome analysis of MeJA and S. delphinii treatment showed exogenous MeJA changed most of the expression of the above genes, and several key members, including DcJAZ1/2/5 and DcMYC2b, are involved in enhancing defense ability to S. delphinii in D. catenatum. CONCLUSIONS: The findings indicate exogenous MeJA treatment affects the expression level of DcJAZ1/2/5 and DcMYC2b, thereby enhancing D. catenatum resistance to S. delphinii. This research would be helpful for future functional identification of core JA pathway genes involved in breeding for disease resistance in D. catenatum.

Amycolatopsis dendrobii sp. nov., an endophytic actinomycete isolated from Dendrobium heterocarpum Lindl.

Three novel actinomycete strains, designated as DR6-1T, DR6-2 and DR6-4, isolated from the roots of Dendrobium heterocarpum Lindl in Thailand were studied using a polyphasic taxonomic approach. The strains grew at 20-37 °C, at pH 5-10 and with 5 % (w/v) NaCl. They contained meso-diaminopimelic acid in the cell-wall peptidoglycan and MK-9(H4) was a major menaquinone. Arabinose and galactose were the major sugars in the cell wall. The predominant cellular fatty acids were iso-C16 : 0 and iso-C15 : 0. The detected polar lipids were diphosphatidylglycerol, hydroxyphosphatidylethanolamine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylglycerol. Strains DR6-1T, DR6-2 and DR6-4 shared 99.9-100 % 16S rRNA gene sequence similarity and were closely related to Amycolatopsis echigonensis JCM 21831T (98.7-98.8%). The approximate genome size of strain DR6-1T was 9.6 Mb with a G+C content of 69.6 mol%. The ANIb and dDDH values between genomic sequences of strain DR6-1T and Amycolatopsis echigonensis JCM21831T, Amycolatopsis rubida JCM 10871T and Amycolatopsis nivea KCTC 39515T were 90.55, 92.25, 92.60%, and 47.20, 52.10 and 52.50%, respectively. Based on the phenotypic, chemotaxonomic and genotypic characteristics, it has been concluded that strains DR6-1T, DR6-2 and DR6-4 represent a novel species of the genus Amycolatopsis for which the name Amycolatopsis dendrobii sp. nov. is proposed. The type strain is DR6-1T (=JCM 33742T=KCTC 49546T=TISTR 2840T).

Quantitative proteomics reveals the antifungal effect of canthin-6-one isolated from Ailanthus altissima against Fusarium oxysporum f. sp. cucumerinum in vitro.

Canthin-6-one, one of the main alkaloid compounds extracted from Ailanthus altissima, has recently attracted increasing interest for its antifungal activity. To evaluate the potential of canthin-6-one in controlling plant fungal diseases, we investigated the antifungal activity of canthin-6-one isolated from A. altissima against Fusarium oxysporum f. sp. cucumerinum (Foc) in vitro. The mycelial growth rate and micro-broth dilution were used to test antifungal activity. Furthermore, label-free quantitative proteomics and parallel reaction monitoring (PRM) techniques were applied to analyze the antifungal mechanism. It was found that canthin-6-one significantly inhibited the growth of Foc, and had higher inhibitory action than chlorothalonil at the same concentration. Proteomic analysis showed that the expression of 203 proteins altered significantly after canthin-6-one treatment. These differentially expressed proteins were mainly involved in amino acid biosynthesis and nitrogen metabolism pathways. These results suggest that canthin-6-one significantly interferes with the metabolism of amino acids. Therefore, it affects nitrogen nutrients and disturbs the normal physiological processes of fungi, and ultimately leads to the death of pathogens. This study provides a natural plant antifungal agent and a new perspective for the study of antifungal mechanisms.

Composition and diversity of bacterial communities in the rhizosphere of the Chinese medicinal herb Dendrobium.

BACKGROUND: Dendrobium is a precious herbal that belongs to Orchidaceae and is widely used as health care traditional Chinese medicine in Asia. Although orchids are mycorrhizal plants, most research still focuses on endophytes, and there is still large amount unknown about rhizosphere microorganisms. To investigate the rhizosphere microbial community of different Dendrobium species during the maturity stage, we used high-throughput sequencing to analyze microbial community in rhizosphere soil during the maturity stage of three kinds of Dendrobium species. RESULTS: In our study, a total of 240,320 sequences and 11,179 OTUs were obtained from these three Dendrobium species. According to the analysis of OTU annotation results, different Dendrobium rhizosphere soil bacteria include 2 kingdoms, 63 phyla, 72 classes, 159 orders, 309 families, 850 genera and 663 species. Among all sequences, the dominant bacterial phyla (relative abundance > 1%) were Proteobacteria, Actinobacteria, Bacteroidetes, Acidobacteria, Firmicutes, Verrucomicrobia, Planctomycetes, Chloroflexi, and Gemmatimonadetes. And through WGCNA analysis, we found the hub flora was also belong to Acidobacteria, Actinobacteria and Proteobacteria. CONCLUSIONS: We found that the rhizosphere bacterial communities of the three kinds of Dendrobium have significant differences, and that the main species of rhizosphere microorganisms of Dendrobium are concentrated in the Proteobacteria, Actinobacteria, and Bacteroidetes. Moreover, the smaller the bacterial level, the greater the difference among Dendrobium species. These results fill knowledge gaps in the rhizosphere microbial community of Dendrobium and provide a theoretical basis for the subsequent mining of microbial functions and the study of biological fertilizers.

Antifeedant and antiphytopathogenic metabolites from co-culture of endophyte Irpex lacteus, phytopathogen Nigrospora oryzae, and entomopathogen Beauveria bassiana.

Five new tremulane sesquiterpenoids were isolated from co-culture of endophyte Irpex lacteus, phytopathogen Nigrospora oryzae, and entomopathogen Beauveria bassiana. All compounds showed obvious antifeedant activities against silkworm with inhibition percentages of 73-99%, at concentrations of 50 µg/cm2. Compound 11 indicated notable antifeedant activity with inhibition percentage of 93% at concentration of 6.25 µg/cm2 among them. Compounds 2, 3, 4, 8, 9, 15 and 16 indicated anti-fungal activities against I. lacteus with MIC values ≤8 µg/mL, compounds 11, 12, 16-18 showed significant anti-fungal activity against N. oryzae with MICs ≤ 4 µg/mL, and compounds 2, 5, 12 and 18 indicated significant anti-fungal activity against B. bassiana with MICs ≤ 8 µg/mL. In addition, the I. lacteus should unite B. bassiana to inhibit the production of phytotoxins from N. oryzae in the ternary culture.

Endophytic fungi, host genotype, and their interaction influence the growth and production of key chemical components of Dendrobium catenatum.

To examine how host plant genotype, endophytic fungal species, and their interaction may affect growth and key chemical content and composition in an important orchid species, we assessed four Dendrobium catenatum cultivars co-cultured with three fungi previously isolated from D. catenatum. Fungal endophytes (Tulasnella sp., Leptosphaeria microscopica, and Guignardia sp.) specifically affected the growth and chemical composition of the four cultivars. Fungal infection significantly increased certain growth traits, especially mid-stem thickness, stem biomass, stem polysaccharide and ethanol-soluble extractive content, and leaf flavonoid and phenol content. Presence or abundance of some key chemical components was also altered by fungal treatment. These increases and alterations were highly dependent on the host genotype. The findings of this study contribute to our understanding of Dendrobium and endophytic fungi interactions, and provide vital information for improving the development and use of endophytic fungi in D. catenatum breeding.

The Cocultured Nigrospora oryzae and Collectotrichum gloeosporioides, Irpex lacteus, and the Plant Host Dendrobium officinale Bidirectionally Regulate the Production of Phytotoxins by Anti-phytopathogenic Metabolites.

The distinctive nature of the endophyte Irpex lacteus, host plant, and the phytopathogen Collectotrichum gloeosporioides resulted in both negative and positive regulation of the production of phytotoxins from Nigrospora oryzae. The coculture of nonhomologous I. lacteus and N. oryzae resulted in a greater number of anti-phytopathogenic metabolites from the dominant endophyte than the coculture of homologous I. lacteus and N. oryzae. The coculture of the phytopathogen N. oryzae and either the nonhomologous (isolation of I. lacteus and N. oryzae from the different plants) or homologous (isolation of I. lacteus and N. oryzae from the same plant) endophyte I. lacteus from different sources indicated that the nonhomologous I. lacteus grew faster than the homologous I. lacteus, and the production of phytotoxic azaphilone from the phytopathogenic N. oryzae decreased due to the inhibition resulting from being cocultured with nonhomologous I. lacteus. On the other hand, the production of phytotoxic azaphilone was promoted by the coculture of two phytopathogens, N. oryzae and C. gloeosporioides. The extract of the host plant, Dendrobium officinale, also increased anti-phytopathogenic metabolite production. Six new phytotoxic azaphilones from N. oryzae, four new tremulane sesquiterpenes from I. lacteus, and a new polyketone were isolated. The endophyte-phytopathogen, phytopathogen-phytopathogen, and endophyte-phytopathogen-host interactions can induce the chemical diversity of novel anti-phytopathogenic metabolites.

An amalgam of pathogenic and beneficial endophytic fungi colonizing four Dendrobium species from Meghalaya, India.

Endophytic fungi are known to play an important role in driving the evolution of plants by conferring adaptational advantages to their host through the production of secondary metabolites and phytohormones. In this study, we evaluated the diversity and phylogenetic relationship of endophytic fungal communities from four Dendrobium species viz., Dendrobium chrysanthum, Dendrobium heterocarpum, Dendrobium hookerianum, and Dendrobium longicornu of Meghalaya, India. A total of 51 culturable endophytic fungi were isolated from the four selected orchid species. The isolates were identified based on nuclear large subunit sequences into 33 species. Approximately 91% of the isolates showed affinity to Ascomycetes, while 9% of the isolates showed BLAST search similarity to Basidiomycetes. The most common genera were Trichoderma and Xylaria. The most prevalent genera were Fusarium, which was detected in all the four Dendrobium species followed by Diaporthe, which was present in three Dendrobium species viz., D. chrysanthum, D. hookerianum, and D. heterocarpum. The Shannon index value of endophytic fungal communities was the highest in D. chrysanthum (2.66), while D. longicornu (1) had the highest Evenness index. The present study revealed that endophytic fungi in these orchids are an amalgam of pathogenic and beneficial fungi, which have, at the least, switched their lifestyle to asymptomatic endophyte in their host. To our knowledge, this is the first such report on the diversity of endophytic fungi in the four selected Dendrobium species from Meghalaya, India.

Molecular Identification of Endophytic Fungi and Their Pathogenicity Evaluation Against Dendrobium nobile and Dendrobium officinale.

: Dendrobium are tropical orchid plants that host diverse endophytic fungi. The role of these fungi is not currently well understood in Dendrobium plants. We morphologically and molecularly identified these fungal endophytes, and created an efficient system for evaluating the pathogenicity and symptoms of endophytic fungi on Dendrobium nobile and Dendrobium officinale though in vitro co-culturing. ReThe colony morphological traits of Dendrobium myco-endophytes (DMEs) were recorded for their identification. Molecular identification revealed the presence of Colletotrichum tropicicola, Fusarium keratoplasticum, Fusarium oxysporum, Fusarium solani, and Trichoderma longibrachiatum. The pathogenicity results revealed that T. longibrachiatum produced the least pathogenic effects against D. nobile protocorms. In seedlings, T. longibrachiatum showed the least pathogenic effects against D. officinale seedlings after seven days. C. tropicicola produced highly pathogenic effects against both Dendrobium seedlings. The results of histological examination of infected tissues revealed that F. keratoplasticum and T. longibrachiatum fulfill Koch's postulates for the existence of endophytes inside the living tissues. The DMEs are cross-transmitted inside the host plant cells, playing an important role in plant host development, resistance, and alkaloids stimulation.

[Isolation and identification of pathogen of Dendrobium officinale gray mold and its prevention and control].

Through investigation,it was found that the main disease of leaves was grey mold on Dendrobium officinale in Hubei province,which has a great impact on the yield and quality of D. officinale. The identification of morphological and molecular biological was used to prove that the pathogen was Botrytis cinerea. Through test the effect of 5 plant source fungicides and 4 antibiotic fungicides on mycelial growth of strain HS1,which proved 0. 3% eugenol had the best inhibitory effect,EC50 was 0. 29 mg·L-1,the second was1% osthol and EC50 was 1. 12 mg·L-1,the EC50 of 0. 5% matrine was 9. 16 mg·L-1,the EC50 of the other six fungicides was higher than 10 mg·L-1. The field control effect test proved that 0. 3% eugenol had the best control effect,reaching 89. 44%,secondly for 1%osthole,which was 77. 17%,0. 5% matrine was in the third place with 62. 37% of effective rate. However,the control effect of the other fungicides was less than 60%. The three plant-derived fungicides were safe for the produce of D. officinale and showed no phytotoxicity. The effect of these fungicides on the growth of D. candidum was tested,and proved that all the fungicides were safe and harmless to D. candidum. This study provides a research basis for the safe and effective prevention and control gray mold of D. officinale.

Diversity of culture-independent bacteria and antimicrobial activity of culturable endophytic bacteria isolated from different Dendrobium stems.

Dendrobium is known for its pharmacological actions including anti-cancer effect, anti-fatigue effect, gastric ulcer protective effect, and so on. At present, only studies on endophytic fungi of Dendrobium affecting the metabolites of host plants have been reported, very little research has been done on endophytic bacteria. In this study, we have demonstrated the great diversity of endophytic bacteria in 6 Dendrobium samples from different origins and cultivars. According to the results of the culture-independent method, the endophytic bacterial community in Dendrobium stems showed obvious different in the 6 samples and was influenced by origin and cultivar. Some bacteria including Ralstonia, Comamonas and Lelliottia were first detected in Dendrobium in this study. Based on the culture-dependent method, a total of 165 cultivable endophytic bacteria isolates were isolated from the sterilized Dendrobium stems, and were classified into 43 species according to the 16S rRNA gene sequence analysis. Moreover, 14 of the 43 strains showed antimicrobial activity against phytopathogen using the Kirby-Bauer method. Strain NA-HTong-7 (Bacillus megaterium, 99.12%) showed the highest antimicrobial activity. This study was the first comprehensive study on endophytic bacteria of Dendrobium from different origins and cultivars, which provides new insights into the endophytic bacteria from Dendrobium.