Sreening And Identification Of Endophytic Actinomycetes From Maize And Research On The Mechanism Of Antigungal Activity Against Exserohilum Turcicum

Maize (Zea mays L.) is an important food crops and fodder, with acreage ranking third in the world after wheat and rice. Acreage of maize in China is also great and wide distributed, and maize is one of the main food in North China, Southwest mountainous area and other wadis region. Currently, a variety of diseases often occur mixed under artificial cultivation conditions, making serious harm that ranges from production cuts to total destruction, being the main factor restricting the development of corn industry. Chemical pesticides are often used for maize disease control traditionally, but chemical pesticides can cause problems such as environmental pollution, pesticide residue and so on that the use of chemical pesticides should be cautious. Efficient, environmentally friendly and safe integrated control measures against plant diseases are the need of today’s social development, and using endophytic antagonistic bacteria to produce biological pesticides can meet this requirement. We studied the population of maize endogenous actinomyces, and revealed their distribution on a macro level, then constructed the maize endogenous actinomycetes library, hoping to discover antagonistic endogenous actinomycetes against common corn soil-borne diseases and lay a foundation for the biological control of maize diseases by studing their inhibitory mechanism. Therefore, we use the healthy corn plants near infected corn plants as the source to isolate endogenous actinomycetes. We identified the novel strains and evaluated the antifungal activity of the isolated strains against Exserohilum turcicum, Helminthosporium maydis, Curvularia lunata and Rhizoctonia solani, and the in vitro and in vivo activity to determinate their correlation, results are as follows:(1) A total of89endophytic actinomycete strains were isolated from healthy maize plants.16S rRNA gene sequence analysis and morphological, physiological and biochemical taxonomic identification demonstrated that NEAU-M9, NEAU-M35and NEAU-M89are three novel actinomycetes, with NEAU-M9a member of the genus Actinoplanes, NEAU-M35a member of the genus Streptomyces and NEAU-M89was a member of the genus Micromonospora.(2) Characterization of strain NEAU-M9using a polyphasic approach. Phylogenetic analysis based on16S rRNA gene sequences suggested that strain NEAU-M9belongs to the genus Actinoplanes. The cell walls of strain NEAU-M9were determined to contain meso-diaminopimelic acid and glycine. Ribose, galactose, rhamnose and mannose were detected as whole-cell sugars. The predominant menaquinones were determined to be MK-9(H4)(59.75%) and MK-9(H6)(40.25%). The phospholipids detected were phosphatidyl ethanolamine (PE) and phosphatidylinositol (PI). The genome G+C content was67.90mol%. On the basis of the morphological, chemotaxonomic characteristics and phylogenetic analysis, strain NEAU-M9is considered to represent a novel species of a new genus within the family Actinoplanes, for which the name Actinoplanes hulinensis sp. nov. is proposed. The type strain of Actinoplanes hulinensis is strain NEAU-M9(=CGMCC4.7036T=DSM45728T)(3) Characterization of strain NEAU-M35using a polyphasic approach. Phylogenetic analysis based on16S rRNA gene sequences suggested that strain NEAU-M35belongs to the genus Streptomyces. The cell walls of strain NEAU-M35were determined to contain L-diaminopimelic acid and glycine. Ribose and galactose were detected as whole-cell sugars. The pedominant menaquinones were MK-9(H4)(53.13%), MK-9(H6)(38.28%) and MK-9(H8)(8.59%). The phospholipids detected were phosphatidyl ethanolamine (PE) and phosphatidylinositol (PI). The DNA G+C content was69.90mol%. Basis on the morphological, chemotaxonomic and phylogenetic analysis, strain NEAU-M35is considered to represent a novel species of the genus Strepomyces, for which the name Streptomyces xiangfangensis sp. nov. is proposed. The type strain of Streptomyces haerbing is strain NEAU-M35(=CGMCC4.7083T=DSM45732T)(4) Characterization of strain NEAU-M89using a polyphasic approach. Phylogenetic analysis based on16S rRNA gene sequences suggested that strain NEAU-M89fell within the family Micromonospora. The cell walls of strain NEAU-M89were determined to contain/weso-diaminopimelic acid and glycine. Xylose, galactose were detected as whole-cell sugars. The major menaquinones were determined to be MK-10(H2)(42.3%), MK-10(H4)(37.2%), MK-8(Hg)(13.7%) and MK-9(H4)(6.8%). The phospholipids detected were diphosphatidylglycerol (DPG), phosphatidyl methyl ethanolamine (PME), phosphatidyl ethanolamine (PE) and phosphatidylinositol (PI). The DNA G+C content was68.80mol%. On the basis of the morphological, chemotaxonomic characteristics and phylogenetic analysis, strain NEAU-M89is considered to represent a novel species of a new genus within the family Micromonospora, for which the name Micromonospora zeae sp. nov. is proposed. The type strain of Micromonospora zeae is strain NEAU-M89(=CGMCC4.7092T=DSM45882T)(5) The in vitro activity of the isolated89strains against four corn pathogenic fungi was determined by the plate confrontation method and criss-cross method, respectively. The results showed that18strains have different degrees of activity against Exserokilum turcicum, Helminthosporium maydis, Curvularia lunata and Rhizoctonia solani. In vitro activity evaluation showed that Micromonospora zeae NEAU-M89has significant antifungal activity against four pathogenic fungi, especially against Exserohilum turcicum and Helminthosporium maydis, with inhibition ratio up to80%. But due to the soil type, cultivation way, pH value and complexity of soil microorganism, the activity of strain NEAU-M89was unstable. In vivo activity of strain NEAU-M89against Exserohilum turcicum was tested using greenhouse pot experiment, and the result showed good in vivo activity of strain NEAU-M89values of control maize.(6) The effect on mycelial growth, spore germination, cell membrane permeability, soluble sugar, phosphorus metabolism and other aspects of the methanol extract of strain NEAU-M89against Exserohilum turcicum was studied to investigate the inhibitory mechanism of strain NEAU-M89against Exserohilum turcicum.Safe, environmentally friendly and efficient integrated measures to control crop diseases is the need of today’s social development, and biological pesticides produced by plant antagonistic endophytic actinomycetes can meet this requirement. Maize endophytic actinomycetes of is a far from fully developed resource with huge potential applications. Identification of maize endophytic actinomycetes can enrich maize endophytic actinomycetes resources and lay a foundation for screening of maize antagonistic endophytic actinomycetes and biological control of maize diseases.