J. Microbiol. Biotechnol. (2014), 24(9), 1149–1161 http://dx.doi.org/10.4014/jmb.1402.02035 Research Article Review jmb Diversity of Endophytic Fungi from Different Verticillium-Wilt-Resistant Gossypium hirsutum and Evaluation of Antifungal Activity Against Verticillium dahliae In Vitro Zhi-Fang Li†, Ling-Fei Wang†, Zi-Li Feng, Li-Hong Zhao, Yong-Qiang Shi, and He-Qin Zhu* State Key Laboratory of Cotton Biology, Institute of Cotton Research of Chinese Academy of Agricultural Sciences, Anyang, Henan 455000, P. R. China Received: February 18, 2014 Revised: May 16, 2014 Cotton plants were sampled and ranked according to their resistance to Verticillium wilt. In Accepted: May 16, 2014 total, 642 endophytic fungi isolates representing 27 genera were recovered from Gossypium hirsutum root, stem, and leaf tissues, but were not uniformly distributed. More endophytic fungi appeared in the leaf (391) compared with the root (140) and stem (111) sections. First published online However, no significant difference in the abundance of isolated endophytes was found among May 19, 2014 resistant cotton varieties. Alternaria exhibited the highest colonization frequency (7.9%), *Corresponding author followed by Acremonium (6.6%) and Penicillium (4.8%). Unlike tolerant varieties, resistant and Phone: +86-372-2562280; susceptible ones had similar endophytic fungal population compositions. In three Fax: +86-372-2562280; Verticillium-wilt-resistant cotton varieties, fungal endophytes from the genus Alternaria were E-mail: [email protected] most frequently isolated, followed by Gibberella and Penicillium. The maximum concentration † These authors contributed of dominant endophytic fungi was observed in leaf tissues (0.1797). The evenness of stem equally to this work. tissue endophytic communities (0.702) was comparatively more uniform than the other two tissues. Eighty endophytic fungi selected from 27 genera were evaluated for their inhibition activity against highly virulent Verticillium dahliae isolate Vd080 in vitro. Thirty-nine isolates exhibited fungistasis against the pathogen at varying degrees. Seven species, having high growth inhibition rates (≥75%), exhibited strong antifungal activity against V. dahliae. The antifungal activity of both volatile and nonvolatile metabolites was also investigated. The nonvolatile substances produced by CEF-818 (Penicillium simplicissimum), CEF-325 (Fusarium solani), CEF-714 (Leptosphaeria sp.), and CEF-642 (Talaromyces flavus) completely inhibited V. dahliae growth. These findings deepen our understanding of cotton-endophyte interactions pISSN 1017-7825, eISSN 1738-8872 and provide a platform for screening G. hirsutum endophytes with biocontrol potential. Copyright© 2014 by The Korean Society for Microbiology Keywords: Cotton, endophytic fungi, V. dahliae, Verticillium wilt, biological control and Biotechnology Introduction include any organism that lives in plant tissue whether neutral, beneficial, or detrimental [43]. Based on their Endophytes are microorganisms that inhabit healthy nature, endophytes can be divided into three categories: (i) plant tissues without causing any apparent or detectable nonpathogenic to own host but are pathogens of other symptoms in the host [39]. The topographical term was hosts; (ii) nonpathogenic microbes; and (iii) pathogens that expanded to include fungi, bacteria, and actinomycetes, have been rendered nonpathogenic but are still capable of which spend either the whole or a period of their life cycle colonization by selection methods or genetic alteration [5]. colonizing the symplastic or apoplastic spaces of In most cases, the relationship between an endophyte and asymptomatic living plant tissues [55]. However, the the host is neither pathogenic nor simply mutualistic [51, definition of an endophyte has been broadened and can 55]; however, host plants sometimes benefit from co- September 2014 ⎪ Vol. 24⎪ No. 9 1150 Li et al. existing endophytes that provide resistance to biotic and has been carried out on either their distribution in tissues abiotic stresses [19, 27]. Specifically, fungal endophytes can or variability in resistance. Far less work has been directly inhibit pathogen infection and proliferation within conducted on the evaluation of their biocontrol potential. the host via antibiosis, competition, and mycoparasitism [6, The objectives of this study were (i) to isolate and identify 36], or indirectly by inducing resistance responses intrinsic the fungi that inhabit the healthy internal tissues from to the host against the fungal pathogens [7]. cotton varieties of varying resistance to Verticillium wilt; Cotton is one of the most important natural fiber crop (ii) to clarify the distribution patterns of endophytic fungi plants worldwide, with a high economic value. Verticillium in different tissues and cotton varieties and calculate the wilt disease, caused by the phytopathogenic fungus colonization frequency and diversity indices; and (iii) to Verticillium dahliae Kleb., is the major threat to cotton screen endophytic fungi with high-level inhibitory activity production [24, 56]. V. dahliae hyphae grow from the root and evaluate their potential as V. dahliae biocontrol agents. surface toward the cortical tissue, which is adjacent to the This work is the first systematic report on the incidence of stele, and subsequently attack the aerial parts of the plant, endophytic fungi from G. hirsutum and their activity resulting in systematic disease [11]. This vascular disease of against the destructive cotton pathogen V. dahliae. cotton cannot be prevented using cultivars or cultural practices alone [42]. Moreover, traditional methods of Materials and Methods chemically controlling these diseases can be expensive and ineffective, and have a negative impact on both Collection of Cotton Varieties environmental and human health. Biological control as Twelve varieties ranked in accordance to Verticillium wilt part of integrated pest management has been suggested as resistance were selected. Jimian 11, Zhongmiansuo 8, and the most sustainable long-term solution [10]. As a new Zhongmiansuo 24 are V. dahliae susceptible, whereas Zhongzhimian source of biological control agents, the use of cotton fungal 2, Glucanase-Chitinase transgenic cotton, and Haidaomian are resistant. The other six varieties formed the tolerant group endophytes for combating Verticillium wilt disease requires (Table 1). All of the cotton plants were cultivated in Anyang, further investigation. Henan Province, China. At the flowering stage, five healthy plants Previous studies have reported on the diversity of the of each variety were collected and separated into leaves, stems, mycoflora community in flowers, the incidence of Fusarium and roots. species associated with roots, and the recovery frequency of fungal endophytes isolated from the stems of native Source of V. dahliae Strain Gossypium species [1, 51, 57]. The endophytic fungal The strongly virulent defoliating V. dahliae isolate Vd080 diversity of transgenic (expresses the Cry1Ac protein) and recovered from Verticillium-wilt-symptomatic cotton in Hebei isoline cotton exhibits consistent patterns in the leaves, Province, China was used for the antagonism test. The isolate was stems, and roots. However, the tissue type and developmental single-spore purified for three generations and stored at -70°C in stage significantly affect the fungal community composition 15% glycerol for long-term preservation and kept at 4°C for 2 days [50]. A few cotton endophytes have been identified as before use. plant-growth-promoting and biocontrol agents. Cladorrhinum Surface Sterilization and Isolation of Endophytic Fungi foecundissimum, an endophytic fungus obtained from cotton The root, stem, and leaf segments were washed in flowing tap stem tissue, colonizes the root tissues and accelerates the water to remove epiphytes and soil debris. The stem and root level of phosphorus uptake and plant growth [18]. The tissues were then rinsed in 75% ethanol for 3 min, and in 5% cotton endophytic fungus Lecanicillium lecanii readily NaClO for 1 min, and then washed three times with sterile water. colonized two potential hosts and was transferred between The leaves were rinsed for 2 min in 75% ethanol, followed by aphids and cotton plants, killing aphids but not causing 30 sec of 5%-NaClO and sterile water washing. obvious disease in cotton leaves [3]. According to Wang et After surface drying, each segment was cut into 0.5 × 0.5 cm2 al. [51], none of the endophytic fungal isolates recovered pieces with a sterile blade. Fifty pieces of each tissue from the from cotton induced foliar symptoms by either root different cotton varieties were placed onto PDA plates (five pieces dipping or stem puncturing inoculation. This finding in each plate) amended with 200 ml/l streptomycin (to prevent supports the theory that Gossypium endophytic fungi are bacterial contamination). To validate the effects of surface sterilization, the suspension collected from the last rinsing step unlikely sources of cotton pathogens. was plated onto PDA medium. To test the sterility of the bench, an Although there are a few fragmentary reports on uncovered blank PDA plate was placed on it for 30 min and then G. hirsutum fungal endophytes, no comprehensive work incubated at 25ºC for 2 days. The tissues were observed for fungal J. Microbiol. Biotechnol. Cotton Fungal Endophyte Diversity and Biocontrol Potential Evaluation 1151 Table 1. The number of endophytic fungi isolated from the tissues of