Scholarly Work - Plant Sciences & Plant Pathology
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Item Genomic and Functional Characterization of the Endophytic Bacillus subtilis 7PJ-16 Strain, a Potential Biocontrol Agent of Mulberry Fruit Sclerotiniose(2018-09) Xu, Wei-Fang; Ren, Hui-Shuang; Ou, Ting; Lei, Ting; Wei, Jun-Hong; Huang, Chuan-Shu; Li, Tian; Strobel, Gary A.; Zhou, Ze-Yang; Xie, JieBacillus sp. 7PJ-16, an endophytic bacterium isolated from a healthy mulberry stem and previously identified as Bacillus tequilensis 7PJ-16, exhibits strong antifungal activity and has the capacity to promote plant growth. This strain was studied for its effectiveness as a biocontrol agent to reduce mulberry fruit sclerotiniose in the field and as a growth-promoting agent for mulberry in the greenhouse. In field studies, the cell suspension and supernatant of strain 7PJ-16 exhibited biocontrol efficacy and the lowest disease incidence was reduced down to only 0.80%. In greenhouse experiments, the cell suspension (1.0 × 106 and 1.0 × 105 CFU/mL) and the cell-free supernatant (100-fold and 1000-fold dilution) stimulated mulberry seed germination and promoted mulberry seedling growth. In addition, to accurately identify the 7PJ-16 strain and further explore the mechanisms of its antifungal and growth-promoting properties, the complete genome of this strain was sequenced and annotated. The 7PJ-16 genome is comprised of two circular plasmids and a 4,209,045-bp circular chromosome, containing 4492 protein-coding genes and 116 RNA genes. This strain was ultimately designed as Bacillus subtilis based on core genome sequence analyses using a phylogenomic approach. In this genome, we identified a series of gene clusters that function in the synthesis of non-ribosomal peptides (surfactin, fengycin, bacillibactin, and bacilysin) as well as the ribosome-dependent synthesis of tasA and bacteriocins (subtilin, subtilosin A), which are responsible for the biosynthesis of numerous antimicrobial metabolites. Additionally, several genes with function that promote plant growth, such as indole-3-acetic acid biosynthesis, the production of volatile substances, and siderophores synthesis, were also identified. The information described in this study has established a good foundation for understanding the beneficial interactions between endophytes and host plants, and facilitates the further application of B. subtilis 7PJ-16 as an agricultural biofertilizer and biocontrol agent.Item VOC profile of endophytic fungi is altered by nature of lignocellulosic biomass feedstock(2014) Eizadora, Tran-Gyamfi Y.; Strobel, Gary A.; Taatjes, Craig A.; Hadi, Masood Z.Item The Production of 1,8-Cineole, a Potential Biofuel, from an Endophytic Strain of Annulohypoxylon sp. FPYF3050 When Grown on Agricultural Residues(2017) Wang, K. Y.; Strobel, Gary A.; Yan, D.-H.An endophytic fungus producing 1,8-cineole from Neolitsea pulchella (Meissn.) Merr. was identified as Annulohypoxylon sp. by phylogenetic analyses of the sequence alignments of ITS rDNA, β-tubulin, Actin and EF1-α. This isolate produces an attractive spectrum of volatile organic compounds (VOCs) with only one dominant component, 1,8-cineole, as identified by gas chromatography-mass spectrometry (GC-MS). The fungus was able to grow in seven media with different carbon sources, and five raw agro-forest residues. The content of 1,8-cineole in the mixed VOCs via fungus reached up to 94.95% and 91.25% relative area in PDA and raw poplar sawdust, respectively. Under optimum test conditions, the fungus produced 1,8-cineole at the 0.764 ppmv in 50 mL head spaces in PDA. Interestingly, 1,8-cineole is an ideal fuel additive for both diesel and gasoline engines. Also, this is the first isolate, in this group of fungi, making cineole, which produces as its primary VOC product which makes it an ideal organism for strain improvement. Such as step will be critical for its ultimate use in biofuel production.Item An Endophytic Nodulisporium sp. from Central America Producing Volatile Organic Compounds with Both Biological and Fuel Potential(2013) Hassan, Syed R.-U.; Strobel, Gary A.; Geary, Brad; Sears, JoeA Nodulisporium sp. (Hypoxylon sp.) has been isolated as an endophyte of Thelypteris angustifolia (Broadleaf Leaf Maiden Fern) in a rainforest region of Central America. It has been identified both on the basis of its morphological characteristics and by scanning electron microscopy as well as ITS sequence analysis. The endophyte produces volatile organic compounds (VOCs) that have both fuel (mycodiesel) and use for biological control of plant disease. When grown on potato dextrose agar, the organism uniquely produces a series of ketones, including acetone; 2-pentanone; 3-hexanone, 4-methyl; 3-hexanone, 2,4- dimethyl; 2-hexanone, 4-methyl, and 5-hepten, 2-one and these account for about 25% of the total VOCs. The most abundant identified VOC was 1,8 cineole, which is commonly detected in this group of organisms. Other prominent VOCs produced by this endophyte include 1-butanol, 2- methyl, and phenylethanol alcohol. Moreover, of interest was the presence of cyclohexane, propyl, which is a common ingredient of diesel fuel. Furthermore, the VOCs of this isolate of Nodulisporium sp. were selectively active against a number of plant pathogens, and upon a 24 h exposure caused death to Phytophthora palmivora, Rhizoctonia solani, and Sclerotinia sclerotiorum and 100% inhibition to Phytophthora cinnamomi with only slight to no inhibition of the other pathogens that were tested. From this work, it is becoming increasingly apparent that each isolate of this endophytic Nodulisporium spp., including the Daldina sp. and Hypoxylon spp. teleomorphs, seems to produce its own unique set of VOCs.Item In vitro antimicrobial effect of the volatile organic compounds from Muscodor crispans against the human pathogenic oomycete- Pythium insidiosum(2012-11) Krajaejun, Theerapong; Lowhnoo, Tassanee; Rujirawat, Thidarat; Yingyong, Wanta; Fucharoen, Suthat; Strobel, Gary A.Pythium insidiosum is an oomycete capable of causing a life-threatening disease in humans, called pythiosis. Conventional antifungal drugs are ineffective against P. insidiosum infection. A synthetic mixture of the volatile organic compounds (VOCs) from the endophytic fungus Muscodor crispans strain B23 demonstrates antimicrobial effects against a broad range of human and plant pathogens, including fungi, bacteria, and oomycetes. We studied the in vitro effects of B23 VOCs against 25 human, 1 animal, and 4 environmental isolates of P. insidiosum, compared with a no-drug control. The B23 synthetic mixture, at amounts as low as 2.5 µl, significantly reduced growth of all P. insidiosum isolates by at least 80%. The inhibitory effect of the B23 VOCs was dose-dependent. The growth of all isolates was completely inhibited by a dose of 10.0 µl of B23 VOCs, and all isolates were killed by a dose of 20.0 µl. Synthetic B23 VOCs of M. crispans had inhibitory and lethal effects against all P. insidiosum isolates tested. Further studies are needed to evaluate this mixture for treatment of pythiosis.Item The Emergence of Endophytic Microbes and Their Biological Promise(2018-05) Strobel, Gary A.As is true with animal species, plants also have an associated microflora including endophytes as well as microbes associated with the phyllosphere and rhizosphere (plant surfaces) and this is considered the plant microbiome. However, those organisms within virtually all tissues and organs of the plant are known as endophytes. Most often fungi are the most frequently recovered endophytes from plant tissues, but bacterial forms generally occur in greater numbers, but not in species varieties. The exact biological/biochemical role of the endophyte in the plant and how it interacts with the plant and other endophytes and plant associated organisms has not been intensely and carefully examined. However, this has not stopped investigators in exploring the direct utility of endophytes in boosting plant production, and discovering that endophytes can directly influence the plant to resist temperature extremes, drought, as well as the presence of disease causing organisms. Also, because of the relationships that endophytes seem to have with their host plants, they make a myriad of biologically active compounds some of which are classified as antibiotics, antioxidants, anticancer agents, volatile antimicrobial agents, immunosuppressive compounds, plant growth promoting agents, and insecticides. These endophytic compounds represent a wide range of organic molecules including terpenoids, peptides, carbohydrates, aromatics, hydrocarbons and others and it seems that these compounds may have a role in the host microbe relationship. Most recently and quite surprisingly, some endophytes have been discovered that make hydrocarbons of the types found in diesel and gasoline fuels. In addition, recently discovered are epigenetic factors relating to the biology and biochemistry of endophytes. Interestingly, only about 1–2% of the entire spectrum of 300,000 known plants have been studied for their endophyte composition. Additionally, only a few plants have ever been completely studied including all tissues for the microbes within them. Likewise, the vast majority of plants, including those in oceans and lower plant forms, have never been examined for their endophytes. Furthermore, endophytes representing the “microbiome” of world’s major food plants as they exist in their native “centers of origin” are largely unknown. This non-classical review is intended to provide background information on aspects of developments in endophyte biology and more importantly the identification of new questions in this field that need to be addressed. The review is primarily based on the author’s long held experience in this field.Item Antifungal Activities of Volatile Secondary Metabolites of Four Diaporthe Strains Isolated from Catharanthus roseus(2018-05) Yan, Dong-Hui; Song, Xiaoyu; Li, Hongchang; Luo, Tushou; Dou, Guiming; Strobel, Gary A.Four endophytic fungi were isolated from the medicinal plant, Catharanthus roseus, and were identified as Diaporthe spp. with partial translation elongation factor 1-alpha (TEF1), beta-tubulin (TUB), histone H3 (HIS), calmodulin (CAL) genes, and rDNA internal transcribed spacer (ITS) region (TEF1-TUB-HIS--CAL-ITS) multigene phylogeny suggested for species delimitation in the Diaporthe genus. Each fungus produces a unique mixture of volatile organic compounds (VOCs) with an abundant mixture of terpenoids analyzed by headspace solid-phase microextraction (SPME) fiber-GC/MS. These tentatively-detected terpenes included α-muurolene, β-phellandrene, γ-terpinene, and α-thujene, as well as other minor terpenoids, including caryophyllene, patchoulene, cedrene, 2-carene, and thujone. The volatile metabolites of each isolate showed antifungal properties against a wide range of plant pathogenic test fungi and oomycetes, including Alternaria alternata, Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium graminearum, and Phytophthora cinnamomi. The growth inhibition of the pathogens varied between 10% and 60% within 72 h of exposure. To our knowledge, the endophytic Diaporthe-like strains are first reported from Catharanthus roseus. VOCs produced by each strain of the endophytic Diaporthe fungi were unique components with dominant monoterpenes comparing to known Diaporthe fungal VOCs. A discussion is presented on the inhibitive bioactivities of secondary metabolites among endophytic Diaporthe fungi and this medicinal plant.Item Halophytes-associated endophytic and rhizospheric bacteria: diversity, antagonism and metabolite production(2018-02) Bibi, Fehmida; Strobel, Gary A.; Naseer, Muhammad I.; Yasir, Muhammad; Khalaf Al-Ghamdi, Ahmed A.; Azhar, Esam I.In Saudi Arabia, halophytes occupy tidal and intertidal forest ecosystems. They and their associated microflora have immense potential to yield novel and important useful natural products. Three halophytes (Avicennia marina, Halocnemum strobilaceum, Zygophyllum qatarense) were targeted for the isolation and identification of populations of endophytic and rhizospheric bacteria having antimicrobial potential. A total 554 bacterial isolates were initially screened against oomycetes fungal pathogens, Phytophthora capsici and Pythium ultimum. Of these, only 57 rhizospheric and endophytic bacteria exhibited inhibition against the targeted bioassay oomycetes. Tentative identification of the bacteria was on the basis of 16S rRNA gene sequences which revealed 92-100% sequence identity to type strains of related species and placed these organisms in six major classes: Actinobacteria, γ-Proteobacteria, Firmicutes, α-Proteobacteria, Flavobacteriia and [beta]-Proteobacteria. When checked for lytic enzyme production, mostly the isolates of Actinobacteria and Firmicutes were potential enzyme producers. Detection of secondary metabolite biosynthetic genes - type I polyketide synthases, type II polyketide synthases and nonribosomal peptide synthetases - confirmed that 21 (35.5%) isolates were positive for at least one type of the biosynthetic gene. In order to identify metabolites, three isolates, Alteromonas australica (EA73), Aidingimonas halophila (EA105) and Halomonas zincidurans (EA127), were selected and subjected to chemical analyses using liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. Both analyses showed the presence of different bioactive compounds in the culture extracts of isolates some of which are already reported for their diverse biological activities such as 2, 4-Diacetylphloroglucinol. Our results demonstrated that halophytes represent an important source of potentially active bacteria producing antifungal metabolites of medical significance.Item Microbial Flora Associated with the Halophyte-Salsola imbricate and Its Biotechnical Potential(2018-01) Bibi, Fehmida; Strobel, Gary A.; Naseer, Muhammad I.; Yasir, Muhammad; Khalaf Al-Ghamdi, Ahmed A.; Azhar, Esam I.Halophytes are associated with the intertidal forest ecosystem of Saudi Arabia and seemingly have an immense potential for yielding useful and important natural products. In this study we have aimed to isolate and characterize the endophytic and rhizospheric bacterial communities from the halophyte, Salsola imbricata, In addition these bacterial strains were identified and selected strains were further studied for bioactive secondary metabolites. At least 168 rhizspheric and endophytic bacteria were isolated and of these 22 were active antagonists against the oomycetous fungal plant pathogens, Phytophthora capsici and Pythium ultimum. Active cultures were mainly identified with molecular techniques (16S r DNA) and this revealed 95.7–100% sequence similarities with relevant type strains. These microorgansims were grouped into four major classes: Actinobacteria, Firmicutes, β-Proteobacteria, and γ-Proteobacteria. Production of fungal cell wall lytic enzymes was detected mostly in members of Actinobacteria and Firmicutes. PCR screening for type I polyketide synthases (PKS-I), type II polyketide synthases (PKS-II) and nonribosomal peptide synthetases (NRPS) revealed 13 of the 22 strains (59%) were positive for at least one of these important biosynthetic genes that are known to be involved in the synthesis of important antibiotics. Four bacterial strains of Actinobacteria with potential antagonistic activity including two rhizobacteria, EA52 (Nocardiopsis sp.), EA58 (Pseudonocardia sp.) and two endophytic bacteria Streptomyces sp. (EA65) and Streptomyces sp. (EA67) were selected for secondary metabolite analyses using LC-MS. As a result, the presence of different bioactive compounds in the culture extracts was detected some of which are already reported for their diverse biological activities including antibiotics such as Sulfamethoxypyridazine, Sulfamerazine, and Dimetridazole. In conclusion, this study provides an insight into antagonistic bacterial population especially the Actinobacteria from S. imbricata, producing antifungal metabolites of medical significance and characterized taxonomically in future.Item Pantoea agglomerans SWg2 colonizes mulberry tissues, promotes disease protection and seedling growth(2017-10) Xie, Jie; Shu, Ping; Strobel, Gary A.; Chen, Jie; Wei, Junhong; Xiang, Zhonghuai; Zhou, ZeyangMulberry bacterial blight caused by Pseudomonas syringae pv. mori, is one of the most serious diseases on mulberry (Morus alba L.), and threatens sericulture development. An endophytic Pantoea agglomerans SWg2 (referred to as SWg2), originally isolated from roots of healthy mulberry, is extremely inhibitory activity to P. syringae. Greenhouse tests were first conducted to examine the biological control properties of SWg2. A suspension of SWg2, introduced to the host plant, significantly reduced disease symptoms caused by P. syringae. Likewise, when a suspension of SWg2 was sprayed onto plantlets 2 days before inoculation with P. syringae, its disease control efficacy was up to 65.6% even after inoculation with P. syringae 18 days later. Interestingly, the control effect of the traditional agrochemical streptomycin was just 33.2% at 18 days post inoculation (DPI), although it was 55.6% at 9 DPI. Furthermore, SWg2 promoted the growth of mulberry seedlings. The immersion of 107 CFU mL−1 suspension of SWg2 had the most distinctive growth-promoting role since the seed germination rate and radicle length development were enhanced up to 100% and 144.2%, respectively. Based on the green fluorescent protein (GFP) labeling technique and a re-isolation method, it was determined that the SWg2 strain colonized and spread in mulberry seedlings. The bacteria entered seedlings through the areas of emergence of root hairs and cracks in the rhizodermis. Eventually these cells spread to other tissues of the plant with colonization occurring mainly in the intercellular spaces. Furthermore, the GFP-tagged SWg2 strain could be detected in stems and leaves, but the quantity was smaller than that in roots. There appears to be no outward negative effects of SWg2 on treated plants and silkworms feed on them in a normal manner. This work indicates that P. agglomerans SWg2 possesses the biological potential to provide protection of its host against the pathogen of mulberry bacterial blight will at the same time promoting the growth and vigor of the host plant.