Identification of endophytes with biocontrol potential from Ziziphus jujuba and its inhibition effects on Alternaria alternata, the pathogen of jujube shrunken-fruit disease

Jian-Feng Lang; Xue-Liang Tian; Ming-Wang Shi; Long-Xian Ran

doi:10.1371/journal.pone.0199466

Identification of endophytes with biocontrol potential from Ziziphus jujuba and its inhibition effects on Alternaria alternata, the pathogen of jujube shrunken-fruit disease

PLoS One. 2018 Jun 26;13(6):e0199466. doi: 10.1371/journal.pone.0199466. eCollection 2018.

Authors

Jian-Feng Lang^{1

2}, Xue-Liang Tian², Ming-Wang Shi², Long-Xian Ran^{1

3}

Affiliations

¹ College of Forestry, Hebei Agricultural University, Baoding, Hebei, People's Republic of China.
² Department of Plant Protection, Henan Institute of Science and Technology, Xinxiang, Henan, People's Republic of China.
³ Hebei Key Lab of Forest Germplasm Resources and Protection, Baoding, Hebei, People's Republic of China.

Abstract

Endophytic strains were isolated from different parts of a healthy "Dongzao" jujube (Ziziphus jujuba Mill. 'Dongzao') to find biocontrol agents against jujube shrunken-fruit disease caused by Alternaria alternata. The strains were screened using A. alternata strain CN193 as the target pathogen. The nutrient competition for all isolates was studied using the dual culture, and their inhibitive capability was tested by measuring the inhibition width of filter paper disks with filtrate. Influence of filtrate from the selected strains with strong inhibition of mycelial growth on spore germination was studied with hanging drop method on concavity slides. Colonization in the jujube leaves was assayed using a rifampicin-resistant mutant of strain St-zn-34 as the screening marker. Strains were identified based on their morphological, physiological, and biochemical characteristics, 16S rDNA sequencing, and phylogenetic analysis. A total of 81 endophytic strains were isolated from the stems, leaves, flowers, and fruits of winter jujube. Among these isolates, 14 strains showed strong antagonism against A. alternata. Further study showed that the filtrate of strains St-zn-9 and St-zn-34 could inhibit the mycelial growth of A. alternata, and the widths of their inhibition zone reached 6.14±0.03 mm and 8.27±0.09 mm, respectively. However, strain St-zn-34 showed stronger inhibition on spore germination than strain St-zn-9. St-zn-34 could significantly reduce the spore germination rate of A. alternata, and the spore did not germinate at all or the germ tube was very short. A rifampicin resistant-derivative of wild-type strain St-zn-34, which was designated as St-zn-34r, was obtained by transferring the strains to media with stepwise-increased rifampicin. Colonization assays indicated that St-zn-34r could colonize in jujube leaves, and the population of St-zn-34r was 1.2×103 CFU/g FW after inoculation for 30 days. Except for its salt tolerance, St-zn-34 was the closest to those of Bacillus subtilis. Thus, the strain was identified as B. subtilis.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Alternaria / growth & development
Alternaria / physiology*
Alternaria / ultrastructure
Colony Count, Microbial
DNA, Ribosomal / genetics
Endophytes / physiology*
Fermentation
Germination
Pest Control, Biological*
Phylogeny
Plant Diseases / microbiology*
Population Dynamics
Spores, Fungal / physiology
Ziziphus / microbiology*

Substances

DNA, Ribosomal

Grants and funding

This study was supported by the Natural Science Foundation of Hebei Province (C2016204202, http://www.hebstd.gov.cn/) and the Science and Technology Project in Henan Province Department of Education (17A210006, http://www.haedu.gov.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.