葡萄细菌性内生菌对引起炭疽病病菌的特性及生物功效

Characterization and bioefficacy of grapevine bacterial endophytes against causing anthracnose disease.

作者信息

Holkar Somnath K, Bhanbhane Vrushali C, Ghotgalkar Prabhavati S, Markad Harshavardhan N, Lodha Tushar D, Saha Sujoy, Banerjee Kaushik

机构信息

Indian Council of Agricultural Research-National Research Centre for Grapes, Pune, Maharashtra, India.

College of Agri-Business Management, Malegaon, Maharashtra, India.

出版信息

Front Microbiol. 2024 Dec 13;15:1502788. doi: 10.3389/fmicb.2024.1502788. eCollection 2024.

INTRODUCTION

Grapevine ( L.), one of the economically important fruit crops cultivated worldwide, harbours diverse endophytic bacteria (EBs) responsible for managing various fungal diseases. Anthracnose () (Penz.) is one of the major constraints in quality grape production and therefore its management is a major concern among the grape growers.

MATERIALS AND METHODS

Among the 50 EBs isolated from healthy leaf segments from the eight grapevine genotypes, biologically potential 20 EBs were purified and identified based on morphological, and biological characteristics and sequence analysis of 16S rRNA region. The antagonistic activities of EBs against were studied conditions.

RESULTS

The colony morphologies of EBs are white and yellow-coloured colonies, circular to irregular in shape, and entire, and flat margins. Among the 20 purified EBs, 19 isolates were found to be Gram-positive except one i.e., MS2 isolate. The 12 isolates reduced nitrate and 14 isolates produced urease enzyme. The assay revealed that two isolates, SB4 and RF1, inhibited 56.1% and 55.6% mycelial growth of , respectively. Further, the identity of EBs was confirmed through PCR amplification of the 16S rRNA region resulting in ~1400 bp size amplicons. The sequence analysis of representative 15 isolates revealed that 5 EB isolates viz., SB5, CS2, RG1, RF1, C1 were identified as with >99% sequence identity, two EBs viz., SB3, and CS1 were identified as subsp. , two EBs viz., SB1, and CS4 were identified as . The SB2 isolate was identified as sp., whereas SB4 as , TH1 as , TH2 as , CS3 as and MS1 as were identified.

CONCLUSION

The phylogenetic analysis of 16S rRNA sequence revealed eight distinct clades and showed the close clustering of identified species with the reference species retrieved from NCBI GenBank. The current investigation provides the scope for further field evaluations of these endophytic microbes for managing anthracnose disease.

引言

葡萄（Vitis vinifera L.）是全球广泛种植的重要经济水果作物之一，其体内含有多种内生细菌（EBs），这些内生细菌可防治多种真菌病害。炭疽病（Colletotrichum acutatum (Simmonds) Penz.）是优质葡萄生产的主要限制因素之一，因此其防治是葡萄种植者主要关注的问题。

材料与方法

从8种葡萄基因型的健康叶片中分离出50株内生细菌，基于形态学、生物学特性以及16S rRNA区域的序列分析，对其中具有生物学潜力的20株内生细菌进行了纯化和鉴定。研究了内生细菌在不同条件下对尖孢炭疽菌（Colletotrichum acutatum）的拮抗活性。

结果

内生细菌的菌落形态为白色和黄色菌落，形状圆形至不规则，边缘整齐、扁平。在20株纯化的内生细菌中，除1株即MS2菌株外，其余19株均为革兰氏阳性菌。12株菌株能还原硝酸盐，14株菌株产生脲酶。平板对峙试验表明，SB4和RF1这两株菌株分别抑制尖孢炭疽菌菌丝生长56.1%和55.6%。此外，通过对16S rRNA区域进行PCR扩增，扩增产物大小约为1400 bp，从而确认了内生细菌的身份。对15株代表性菌株的序列分析表明，5株内生细菌菌株即SB5、CS2、RG1、RF1、C1被鉴定为胶孢炭疽菌（Colletotrichum gloeosporioides），序列同一性>99%；两株内生细菌菌株即SB3和CS1被鉴定为尖孢炭疽菌（Colletotrichum acutatum）亚种；两株内生细菌菌株即SB1和CS4被鉴定为暹罗炭疽菌（Colletotrichum siamense）。SB2菌株被鉴定为炭疽菌属（Colletotrichum）的一个种，而SB4被鉴定为尖孢炭疽菌（Colletotrichum acutatum），TH1被鉴定为暹罗炭疽菌（Colletotrichum siamense），TH2被鉴定为暹罗炭疽菌（Colletotrichum siamense），CS3被鉴定为胶孢炭疽菌（Colletotrichum gloeosporioides），MS1被鉴定为炭疽菌属（Colletotrichum）的一个种。