Diversity of Endophytic Fungi from Different Verticillium-Wilt-Resistant

Home , Aspergillus aculeatus, Bionectria ochroleuca, Dothideomycetes, Eurotiomycetes, Saccharomycetes, Stemphylium solani

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.

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-

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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

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Table 1. The number of endophytic fungi isolated from the tissues of variably resistant cotton varieties. Tissue type Cotton variety (Abbr.) Resistance levela Total Root Stem Leaf Jimian 11 (JM11) S 7 386 96 Zhongmiansuo 8 (ZM8) S 11 4 14 29 Zhongmiansuo 24 (ZM24) S 16 6 29 51 Zhongmiansuo 35 (ZM35) T 16 9 34 59 Yumian 2067 (YM2067) T 17 9 32 58 PCD transgenic cotton (PCD) T 11 4 32 47 Lumianyan 28 (LM28) T 9 10 28 47 Jimian 668 (JM668) T 17 21 9 47 Lumianyan 21 (LM21) T 7 1317 37 Zhongzhimian 2 (ZZM2) R 15 9 34 58 Glu-Chi transgenic cotton (Glu-Chi) R 11 9 15 35 Haidaomian (HDM) R 314 61 78 Total - 140 111 391 642 aV. dahliae resistance varied among the cotton varieties. S, T, and R indicate susceptible, tolerant, and resistant, respectively. growth on alternate days for 15 days. Actively growing fungal ver. 5.05 after multiple alignment of the data by Clustal_X [46], tips emerging from plant tissues were subcultured on PDA for with gaps treated as missing data. Clustering was performed identification and enumeration. using the neighbor-joining method [41]. Bootstrap analysis was All of the endophytic fungi isolated were assigned specific code used to evaluate the tree topology of the neighbor-joining data by numbers (CEF-001-CEF-842) and maintained on PDA slants at performing 1,000 resamplings [14]. The ITS rDNA sequence was 4ºC. All of the samples were deposited at the Plant Protection deposited in the GenBank nucleotide sequence database under Department, Institute of Cotton Research, CAAS. accession numbers KF998977-KF999032 and KF657726-KF657729.

Molecular Identification of Endophytic Fungi Statistical Analysis Pure endophytic fungi were cultured with agitation in Czapek The colonization frequency (% CF) of fungal species was

Dox liquid medium (NaNO3, 2.00 g; K2HPO4, 1.00 g; KCI, 0.50 g; calculated according to Hata and Futai [23] as follows:

MgSO4·7H2O, 0.50 g; FeSO4, 0.01 g; sucrose, 30.00 g; H2O, 1,000 ml) CF = (Ncol/Nt) × 100, where Ncol = number of segments colonized o at 25 ± 1 C for 7 days. Genomic DNA was extracted from frozen by each fungus, and Nt = total number of segments examined. mycelia following the method described by Al-Samarrai and The isolation recovery (IR) of a single endophytic taxon from Schmid [2]. PCR amplification of the internal transcribed spacer the cotton tissue was calculated according to the following (ITS) region of the fungal DNA was conducted with the following formula: universal fungus-specific primers: ITS1 (5’-TCCGTAGGTGAA IR = Ni/Na × 100, where Ni = number of each fungus recovered CCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) from the given tissue, and Na = total number of fungi isolated [54]. PCR amplifications were performed with a Dongsheng EDC- from the given tissue. 810 thermocycler (Dongsheng Innovation Biotech Co., Ltd., The fungal endophytic diversity of each cotton variety and the Guangdong, China) in a total reaction volume of 50 µl, containing different cotton tissues was estimated with five diversity indices. 1× Taq buffer, 0.5 U of Taq DNA polymerase (Takara, Dalian, The reason for using these diversity indices was to take advantage China), 2 mM dNTPs, 2 µM of each primer, and approximately of the strengths of each index and to predict the complete 25 ng of template DNA. The initial DNA denaturation step was structure of different populations. Simpson’s index of dominance performed at 94°C for 2 min, followed by 30 cycles of (D) was calculated by the following formula [44]: denaturation at 94°C for 1 min, annealing at 55°C for 30 sec, and D=Σ(n/N)2, where n = the total number of isolates of a extension at 72°C for 1min, with a final extension at 72°C for particular species, and N = the total number of isolates of all 10 min. ITS products were sequenced by the GENEWIZ species. Corporation, Suzhou, China. The resulting sequence of the ITS Simpson’s diversity index = 1 − D. rDNA was compared with those available in GenBank using the Species richness = S/ N , where S = total number of species. BLAST network service to determine their phylogenetic Shannon-Wiener index (H’) = −Σ(piInpi), where pi = n/N. affiliation. The phylogenetic analysis was performed using MEGA Species evenness E = H’/InS.

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In Vitro Fungal Endophytic Antagonism Test where R1 is the radius length of the V. dahliae isolate Vd080 as

The antifungal activity of isolated endophytic fungi against the control. For the dual culture antagonistic bioassay, R2 is the radial V. dahliae Vd080 isolate was evaluated by three different methods. distance of Vd080 grown on a line between the inoculation of the These were dual culture and volatile and nonvolatile metabolite pathogen and endophytic fungus. For the volatile and nonvolatile bioassays. Each treatment had three replicates and each metabolite bioassays, R2 is the radial distance of Vd080 grown experiment was conducted twice. with endophytic fungi. An inhibitory effect of more than 75% was considered strong inhibition, 50–75% moderate, 25–50% low, and Dual Culture Antagonistic Bioassay 10–25% slight. The dual culture technique was used to assay the antagonistic 2 activity of endophytic fungal isolates. A plug of 5 mm diameter Results from the edge of an actively growing V. dahliae Vd080 was placed at the periphery of the culture plate and incubated for 4 days at Isolation of Endophytic Fungi from Cotton 2 25 ± 1ºC. The plate was then inoculated with a 5-mm -diameter Endophytic fungi were isolated from asymptomatic root, mycelial disc of the endophytic isolate, placed 5 cm from the stem, and leaf segments of cotton plants following a pathogen at the opposite side, and incubated at 25 ± 1ºC. A plate standard isolation protocol. In total, 642 endophytic fungi inoculated with V. dahliae alone served as a control. Each were obtained from 12 Verticillium-wilt-resistant cotton treatment had three replicates. The growth inhibition rate was calculated. varieties, consisting of three susceptible, three resistant, and six tolerant varieties. The healthy living tissues of Volatile Metabolite Inhibitory Bioassay susceptible cotton plants harbored 176 endophytic fungi The effect of volatile antibiosis of endophytic fungi on Vd080 with an average of 59 per variety. In the three resistant was tested in the assemblage described by Dennis and Webster varieties, 171 fungi were observed in the internal tissues [12] and Naraghi et al. [38]. A 5 mm2 disc of Vd080 was placed on with an average of 57 per variety. In the tolerant varieties, petri dishes containing PDA medium and incubated at 25 ± 1°C 295 endophytic fungi were isolated from six plants with an 2 for 4 days. Then, 5 mm discs of endophytic fungi were also average number of 49 per variety. Of the 12 cotton varieties, cultured on petri dishes containing PDA medium. Two petri dish the susceptible cotton JM11 had the largest population (n = bottoms (without their lids) containing pathogen and antagonist 96). However, ZM8, which was also a susceptible variety, isolates were placed face to face and then sealed with thin plastic carried the least endophytic fungi (n = 29). The abundance film. The control petri dishes were not inoculated with antagonistic of isolated endophytic fungi did not differ significantly fungi. The petri dishes were incubated at 25 ± 1°C. among the resistant cotton varieties (Table 1). Nonvolatile Antibiotic Inhibitory Assay The populations of endophytic fungi associated with The effect of accumulation of nonvolatile antibiotics produced different cotton tissue types were also compared. The by endophytic fungi on Vd080 growth was examined using the distribution of endophytic fungi within cotton was not exudate filtering method [53]. Petri dishes containing PDA uniform. With the exception of JM668, a tolerant variety, medium were completely covered with two layers of autoclaved the number of fungi found in the leaves was greater than in cellophane and centrally inoculated with 5 mm2 discs of endophytic both the roots and stems. Out of 642 endophytic fungi fungus. The plates were incubated for 5–7 days at 25 ± 1°C before isolates, the majority (n = 391, 60.9%) were found in the 2 the cellophane and antagonist were removed. A 5 mm pathogen leaves, followed by the roots (n = 140, 21.8%), and stems (n disc was then placed in the center of the dish. The dishes were = 111, 17.3%) (Table 1). incubated for a further 15 days before radial growth was recorded and compared with that of the control plates. Identification and Distribution of Endophytic Fungi in Growth Inhibition Analysis Different Tissues The Vd080 mycelial colony growth (mm) data was recorded at Based on the ITS phylogenetic analysis, 642 endophytic 2-day intervals. A final observation on radial mycelial colony fungi were divided into 27 groups representing 27 different growth was made after approximately 7–15 days, when the genera, including 60 species. With the exception of Rhizoctonia presence of overlapping fungi on any one petri dish was recorded. solani, which belongs to the phylum Basidiomycota, all of The growth inhibition rate (GI) was calculated using the following the isolates were members of the phylum Ascomycota. formula given by Whipps [52]: This included members of the classes Sordariomycetes, R –R Dothideomycetes, Eurotiomycetes, and Saccharomycetes GI = ------1 -2 ×100 R1 (Fig. 1).

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Fig. 1. ITS-based phylogenetic tree showing the relationships among cotton endophytic fungi and closely related sequences from GenBank. Bootstrap values (n = 1,000 replicates) above 50% are shown. The scale bar represents the number of changes per nucleotide position. Accession numbers are given at the end of each sequence ID.

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Alternaria, a Dothideomycete, exhibited the highest total 20.7%) and Acremonium (CF = 4.0%, IR = 17.1%). Leptosphaeria, colonization frequency (CF = 7.9%). Out of 642 endophytic Trichoderma atroviride, Ascomycete, and Phoma were only fungal isolates, 143 belonged to this group, which isolated from root tissues in this experiment. In stem comprised eight species. Of these, Alternaria alternata was tissues, 19 genera were identified in all. The most common the most common. The second largest endophyte genus genus was Gibberella with the highest colonization was Acremonium, which comprised four species and frequency (4.0%) and isolation recovery (21.7%), followed contained 119 isolates with a CF of 6.6%. Penicillium, which by Penicillium and Acremonium, Rhizoctonia solani, Plectosphaerella, represented the third most dominant group, comprised 87 and Meyerozyma caribbica, which only appeared in stem endophytic fungi and had a CF value of 4.8% (Table 2). tissues. The group Alternaria was the largest population in The endophytic fungi harbored in the cotton roots, stems, the fungal endophytes isolated from cotton leaves (CF = and leaves exhibited a diverse population structure. In root 20.7%, IR = 31.7%). The second most dominant species was tissues, 140 fungal isolates belonging to 19 genera were Trichoderma atroviride with 13.3% CF and 20.5% IR. recovered from 600 segments. Of these, Gibberella exhibited Colletotrichum, Pestalotiopsis, Stemphylium solani, and Magnaporthe the highest colonization frequency (5.2%) and isolation oryzae were found exclusively in cotton leaves (Table 2). recovery (22.5%), followed by Penicillium (CF = 4.8%, IR = The most common species isolated from the roots, stems,

Table 2. The distribution and frequency of endophytic fungi isolated from cotton tissues. Root Stem Leaf Total Endophytic fungus Na IRb CFc N IR CF N IR CF CF Alternaria* 9 6.4 1.5 10 9 1.7 124 31.7 20.7 7.9 Colletotrichum* 0 0 0 0 0 0 9 2.31.5 0.5 Penicillium* 29 20.7 4.8 17 15.32.8 41 10.5 6.8 4.8 Pestalotiopsis sp. 0 0 0 0 0 0 1 0.30.2 0.1 Pichia guilliermondii 2 1.4 0.31 0.9 0.2 16 4.1 2.7 1.1 Stemphylium solani 0 0 0 0 0 0 2 0.5 0.30.1 Verticillium* 4 2.9 0.7 32.7 0.5 1 0.30.2 0.4 Leptosphaeria sp. 1 0.7 0.2 0 0 0 0 0 0 0.1 Chaetomium globosum 32.1 0.5 1 0.9 0.2 2 0.5 0.30.3 Trichoderma atroviride 2 1.4 0.30 0 0 0 0 0 0.1 Acremonium* 24 17.1 4 15 13.5 2.5 80 20.5 13.3 6.6 Aspergillus* 7 5 1.2 4 3.6 0.7 15 3.8 2.5 1.4 Trichothecium roseum 32.1 0.5 1 0.9 0.2 8 2.1 1.30.7 Gibberella* 31 22.5 5.2 24 21.7 4 49 12.3 8.2 5.8 Stachybotrys* 0 0 0 32.7 0.5 1 0.30.2 0.2 Fusarium* 15 10.7 2.5 19 17.1 3.2 31 7.9 5.2 3.6 Cladosporium* 2 1.4 0.32 1.8 0.330.8 0.5 0.4 Dothideomycetes sp. 2 1.4 0.32 1.8 0.30 0 0 0.2 Magnaporthe oryzae 0 0 0 0 0 0 1 0.30.2 0.1 Rhizoctonia solani 0 0 0 2 1.8 0.30 0 0 0.1 Ascomycete sp. 1 0.7 0.2 0 0 0 0 0 0 0.1 Nigrospora oryzae 2 1.4 0.32 1.8 0.30 0 0 0.2 Plectosphaerella sp. 0 0 0 1 0.9 0.2 0 0 0 0.1 Bionectria* 1 0.7 0.2 2 1.8 0.34 1 0.7 0.4 Phoma sp. 1 0.7 0.2 0 0 0 0 0 0 0.1 Meyerozyma caribbica 0 0 0 1 0.9 0.2 0 0 0 0.1 Talaromyces * 1 0.7 0.2 1 0.9 0.2 30.8 0.5 0.3

*At least two species of the genus were found. aThe number of isolates; bisolation recovery; ccolonization frequency.

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Table 3. Colonization frequency (%) of endophytic fungi and leaves were Alternaria, Penicillium, Pichia guilliermondii, common to all three tissue types. Acremonium, Aspergillus, Trichothecium roseum, Bionectria, Genus/Species Root Stem Leaf Chaetomium globosum, Cladosporium, Fusarium, Talaromyces, Alternaria 1.5 1.7 20.7 Gibberella, and Verticillium. The 13 common taxa, except for Penicillium 4.8 2.8 6.8 Verticillium and Chaetomium globosum, isolated from the Pichia guilliermondii 0.30.2 2.7 leaves had a slightly lower colonization frequency than Acremonium 4 2.5 13.3 those from the roots and stems. The other 12 genera or Aspergillus 1.2 0.7 2.5 species recovered from the leaves exhibited higher Trichothecium roseum 0.5 0.2 1.3 colonization frequencies (Table 3). Bionectria 0.2 0.30.7 Chaetomium globosum 0.5 0.2 0.3 Incidence of Fungal Endophytes in Verticillium-Wilt- Cladosporium 0.30.30.5 Resistant Cotton Varieties The population composition of endophytic fungi varied Fusarium 2.5 3.2 5.2 among susceptible, resistant, and tolerant cotton plants. A Talaromyces 0.2 0.2 0.5 total of 176 isolates, representing 19 genera, were isolated Gibberella 5.2 4 8.2 from three susceptible cotton varieties, which were Verticillium 0.7 0.5 0.2

Table 4. The distribution of endophytic fungi in cotton varieties of varying resistance to V. dahliae. Endophytic Susceptible varieties Resistant varieties Tolerant varieties Total Total Total Total fungus JM11 ZM8 ZM24 ZZM2 Glu-Chi HDM ZM35 YM2067 LM28 JM668 LM21 PCD Alternaria*581665162835164655743143 Colletotrichum* 8008 0 0 1 1 0 0 0 0 0009 Penicillium* 10 4 10 24 13 4 9 26 5 9 3 9 4 7 37 87 Pestalotiopsis sp.1001 0 0 0 0 0 0 0 0 0001 Pichia guilliermondii 202 4 0 0 8 8 0 2 0 0 1 4719 Stemphylium solani 200 2 0 0 0 0 0 0 0 0 0 002 Verticillium* 3003 0 0 0 0 5 0 0 00ss058 Leptosphaeria sp.1001 0 0 0 0 0 0 0 0 0001 Chaetomium globosum 111 3 1 0 0 1 0 0 0 1 0 126 Trichoderma atroviride 200 2 0 0 0 0 0 0 0 0 0 002 Acremonium*56172812742313241367568119 Aspergillus* 1 0 1 2 5 1 6 12 3 0 1 2 0 6 12 26 Trichothecium roseum 011 2 2 0 0 2 1 1 3 0 2 1812 Gibberella* 0 9 9 18 13 11 8 32 12 9 9 11 7 6 54 104 Stachybotrys* 0000 1 0 0 1 1 2 0 0 0034 Fusarium* 0 5 3 8 4 3 6 13 2 5 9 10 11 7 44 65 Cladosporium* 0000 0 1 2 3 0 2 0 2 0047 Dothideomycetes sp.0101 2 0 0 2 0 0 0 1 0014 Magnaporthe oryzae 000 0 0 0 0 0 0 0 1 0 0 011 Rhizoctonia solani 000 0 0 0 0 0 0 0 2 0 0 022 Ascomycete sp.0000 0 1 0 1 0 0 0 0 0001 Nigrospora oryzae 200 2 2 0 0 2 0 0 0 0 0 004 Plectosphaerella sp.0000 0 0 0 0 1 0 0 0 0011 Bionectria* 0101 1 0 3 4 0 0 0 0 0227 Phoma sp. 0000 0 1 0 1 0 0 0 0 0001 Meyerozyma caribbica 001 1 0 0 0 0 0 0 0 0 0 001 Talaromyces* 0000 1 0 3 4 0 0 0 0 0115 Total 96 29 51 176 58 35 78 171 59 58 47 47 37 47 295 642

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JM11(96), ZM8(29), and ZM24(51). In JM11, 58 Alternaria Table 5. Diversity indices of endophytic fungi colonizing species accounted for 60.4% of the total isolates. However, cotton root, stem, and leaf tissues. in the ZM8 family, the dominant group was Gibberella, Index Leaf Root Stem accounting for 31%. The endophytic fungi originating from Simpson’s dominance (D) 0.1797 0.1424 0.1308 ZM24 exhibited a different population structure. The Simpson’s diversity index 0.82030.8576 0.8692 biggest group, Acremonium, had 17 isolates, accounting for Species richness 1.416 2.3664 2.6576 33.3%. Shannon-Wiener index (H’) 2.0535 2.274 2.3393 The endophytic fungal population composition of resistant Species evenness (E) 0.61630.6824 0.702 cotton varieties displayed a similar pattern to that of the susceptible ones. Penicillium, Acremonium, and Gibberella were the main groups recovered from ZZM2. Despite the those of the other two tissues. Both Simpson’s and widespread occurrence of the genus Alternaria, only one Shannon-Wiener diversity indices (0.8692, 2.3393) were Alternaria sp. was isolated from ZZM2. Gibberella was the highest in stem tissue endophyte communities. Species most common genus in the transgenic cotton Glu-Chi, and richness was also greatest in stem endophytes (2.6576). The Alternaria represented the main group of endophytic fungi evenness of all communities was also calculated in which recovered from HDM. In three Verticillium-wilt-resistant the stem tissue endophytic community (0.702) was cotton varieties, fungal endophytes of the genus Alternaria comparatively more uniform than the other two tissues were isolated most frequently, followed by Gibberella and (Table 5). Penicillium. Endophytic fungi from the tolerant cotton PCD had the Compared with the susceptible and resistant cotton highest Simpson’s (0.8791) and Shannon-Wiener diversity varieties, there were some notable differences in the indices (2.2045); the lowest endophyte diversity was from representation of some genera across the six tolerant susceptible cotton variety JM11. The ZM8 endophytic varieties. Acremonium was the most dominant group, with community had the greatest species richness (5.1995), the most isolates being recovered from ZM35, YM2067, and followed by ZM24 and LM21. The numbers of individual LM28. The genus Gibberella represented the largest species were most evenly distributed in the PCD endophytic fungal population from JM668. In LM21 and endophytic community (0.6616), followed by ZZM2 and the PCD transgenic cotton varieties, Fusarium was the HDM (Table 6). largest proportion of the endophytic fungal population (Table 4). V. dahliae Inhibitory Activity of Endophytic Fungi A total of 80 endophytic fungi selected from 27 genera Endophytic Community Diversity Analysis were evaluated for their V. dahliae inhibitory activity in The diversity of the endophytic communities isolated vitro. The results of the dual culture antagonistic bioassay from different tissues and cotton varieties was compared showed that endophytes isolated from cotton varied using indices of α-diversity (Simpson's diversity index and greatly in their fungistatic activities against this cotton Shannon-Wiener index) and their components, including pathogen. Nearly half of the endophytes (41 isolates) tested Simpson’s dominance, and species richness and evenness. had no effect on normal V. dahliae growth; the antibiotic The concentration of dominance or Simpson’s dominance ability of the remaining 39 isolates was analyzed further. of endophytic fungi was highest in the leaf tissues (0.1797). Out of 39, seven (17.9%) endophytic fungi exhibited strong A large index value proportionally reflects greater species antifungal activity toward Vd080 with high growth diversity. That is, leaf endophytes were more diverse than inhibition rates (≥75%). CEF-714, which was identified as

Table 6. Diversity indices of endophytic fungi from different cotton varieties. Index JM11 ZM35 YM2067 ZM8 ZZM2 LM28 JM668 LM21 ZM24 Glu-Chi PCD HDM Simpson’s dominance (D) 0.3887 0.1824 0.2354 0.1938 0.1605 0.177 0.1689 0.1936 0.1918 0.2011 0.1209 0.1815 Simpson’s diversity index 0.6113 0.8176 0.7646 0.8062 0.8395 0.823 0.8311 0.8064 0.8082 0.7989 0.8791 0.8185 Species richness 2.8577 3.6453 3.6766 5.1995 3.6766 4.0842 4.0842 4.6032 4.7329 3.9208 4.0842 3.1704 Shannon-Wiener index (H’) 1.5221 1.9029 1.7574 1.846 2.0905 1.9007 1.91931.7569 1.8528 1.8456 2.2045 2.0314 Species evenness (E) 0.4568 0.5711 0.5274 0.554 0.6274 0.5704 0.576 0.52730.556 0.55390.6616 0.6096

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Table 7. The dual cultural antagonistic activity of 39 Leptosphaeria sp., exhibited maximal inhibition ability with endophytic fungal isolates against V. dahliae. the mean growth inhibition rate being 88.1%. V. dahliae GIa growth was also strongly inhibited by CEF-642, Talaromyces Code No. Endophytic fungus Level (Mean ± SD%) flavus (GI = 83.8%). In addition, the isolates CEF-026 CEF-002 Nigrospora oryzae 26.2 ± 1.5 Low (Gibberella intermedia), CEF-193 (Acremonium sp.), CEF-325 CEF-017 Dothideomycetes sp. 30.3 ± 1 Low (Fusarium solani), CEF-818 (Penicillium simplicissimum), and CEF-026 Gibberella intermedia 75.7 ± 4.3Strong CEF-109 (Ascomycete sp.) belonged to this strongly antagonistic group. CEF-421 (Aspergillus aculeatus), CEF-718 (Penicillium CEF-062 Rhizoctonia solani 25 ± 10.7 Slight sp.), CEF-106 (Talaromyces stollii), and CEF-070 (Alternaria CEF-070 Alternaria tenuissima 63.5 ± 3 Moderate tenuissima) comprised a moderately antifungal group with CEF-074 Phoma sp. 33.8 ± 1.6 Low growth inhibition rates ranging from 50% to 75%. The other CEF-100 Chaetomium globosum 33.6 ± 3.3 Low 28 isolates, including 17 with low and 11 with slight growth CEF-106 Talaromyces stollii 58.2 ± 8 Moderate inhibition levels, exhibited reduced fungistatic activity CEF-109 Ascomycete sp. 81.2 ± 1.6 Strong (Table 7). The antagonistic group that comprised seven CEF-117 Acremonium implicatum 24.1 ± 5.6 Slight endophytic cotton fungi was selected to test both volatile CEF-125 Aspergillus oryzae 31.9 ± 3.3 Low and nonvolatile V. dahliae fungistasis. CEF-193 volatile substances exhibited the most V. dahliae CEF-142 Penicillium pinophilum 39.2 ± 5.5 Low inhibition activity, having a growth inhibition rate of CEF-152 Stachybotrys chartarum 34.5 ± 0 Low 94.3%. CEF-642 also strongly reduced the growth of the CEF-160 Alternaria alternata 42.5 ± 1.5 Low targeted pathogen, with an 81.6% inhibition rate. CEF-818, CEF-174 Alternaria porri 32.8 ± 8.2 Low CEF-714, and CEF-325 also produced volatile metabolites CEF-175 Trichothecium roseum 16.4 ± 5.9 Slight that greatly inhibited Vd080 growth (Fig. 2). CEF-193 Acremonium sp. 76.4 ± 2.4 Strong The results of the nonvolatile antibiotic assay revealed CEF-199 Penicillium funiculosum 26.2 ± 1.5 Low that CEF-818, CEF-325, CEF-714, and CEF-642 secreted the CEF-200 Alternaria brassicae 22.4 ± 6 Slight highest amounts of V. dahliae resistant nonvolatile substances. The V. dahliae mycelia did not spread normally CEF-204 Alternaria solani 23.3 ± 9.9 Slight on the PDA plates containing the antibiotic substances CEF-282 Alternaria compacta 20.7 ± 8 Slight mentioned above. The inhibition rate reached to 100%. CEF-325 Fusarium solani 78.0 ± 3.1 Strong V. dahliae growth was moderately suppressed by CEF-193 CEF-332 Gibberella moniliformis 46.8 ± 5.6 Low nonvolatiles. However, the interaction between CEF-026 CEF-340 Aspergillus niger 39.2 ± 11.1 Low CEF-377 Penicillium aculeatum 42.5 ± 8.1 Low CEF-402 Cladosporium sp. 21.9 ± 13.8 Slight CEF-412 Bionectria ochroleuca 39.2 ± 1.6 Low CEF-421 Aspergillus aculeatus 56 ± 2.3Moderate CEF-642 Talaromyces flavus 83.8 ± 3 Strong CEF-646 Plectosphaerella sp. 40.3 ± 4 Low CEF-681 Chaetomium sp. 32.7 ± 7.7 Low CEF-714 Leptosphaeria sp. 88.1 ± 0.2 Strong CEF-716 Trichoderma sp. 24.1 ± 6.3Slight CEF-717 Trichoderma atroviride 22.9 ± 1.6 Slight CEF-718 Penicillium sp. 57.8 ± 2.7 Moderate CEF-764 Cladosporium colocasia 24.1 ± 0 Slight CEF-772 Stemphylium solani 30.2 ± 7.1 Low CEF-818 Penicillium simplicissimum 79.9 ± 2.7 Strong

CEF-833 Alternaria sp. 22.4 ± 4.5 Slight Fig. 2. The volatile antagonistic activity of cotton endophytic aGrowth inhibition rate. fungi against V. dahliae.

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and the number of endophytic fungi inhabiting the cotton varieties (Table 1). This result might be attributed to the fact that all of the cotton plants used in this study were collected from the same field. However, the cotton tissues, collection location, environmental conditions, and plant developmental stage significantly influenced the abundance and composition of the fungal community, but variety did not [48, 51]. Furthermore, no difference was observed between transgenic Bt cotton and its acceptor cotton in terms of endophytic fungi [50]. Endophytic fungi were typically identified through a comparison of morphological features. Distinguishing between closely related or morphologically similar species was a complex task. Furthermore, a large number of endophytic fungi failed to sporulate in culture and could Fig. 3. The nonvolatile antagonistic activity of cotton not be identified, which is a problem commonly reported endophytic fungi against V. dahliae. in the literature [4, 17, 22, 25, 30]. Molecular methods have greatly increased our knowledge regarding fungal diversity nonvolatiles and Vd080 was slight. The inhibition rate was and taxonomy. Several recent studies have shown that only 13.7% (Fig. 3). molecular methods can be successfully used in the study of endophytic fungi [35, 47]. The most widely used gene Discussion regions for the detection of endophytic fungi are the ribosomal DNA sequences, especially the internal transcribed Endophytes inhabit healthy plant tissues, which provide spacer region (ITS1, 5.8S, and ITS2) [9, 34]. Based on the ITS nutrition and shelter. At the same time, they are viewed as characteristics, 642 endophytic cotton fungi were successfully an excellent source of bioactive compounds for the host [31, identified, representing 27 different genera and 60 species. 49]. They also produce many functional metabolites that The G. hirsutum endophytic assemblage is composed of a impact plant health and growth [37]. Of these, some signal number of cosmopolitan genera such as Alternaria, Acremonium, metabolites independently trigger plant defense cascades Penicillium, Gibberella, and Fusarium. The former three as pathogens of other hosts (systemic acquired resistance) genera were the most frequently isolated from G. hirsutum. and nonpathogens (induction of systemic resistance), providing The dominant group was Alternaria with 143 members higher levels of pathogen resistance in plants [5, 40]. comprising eight species. This finding is in accordance It is clear that conducting a comprehensive study of any with a previous report on the endophyte frequency of four host plant is necessary before screening the potential of native Gossypium species in Australia [51]. In addition, their endophytic fungi. In this study, 642 endophytic fungi Alternaria, Gibberella, and Fusarium have been reported as were obtained from the roots, stems, and leaves of 12 the most encountered endophytes in various types of cotton varieties with varying resistance to Verticillium wilt. plants [20, 29]. Thirteen common taxa in this study were In accordance with previous reports [19, 25, 32, 50], the Alternaria, Penicillium, Pichia guilliermondii, Acremonium, number of endophytes recovered from the leaves (391) was Aspergillus, Trichothecium roseum, Bionectria, Chaetomium significantly larger than that from roots and stems. Most globosum, Cladosporium, Fusarium, Talaromyces, Gibberella, leaf endophytes had a greater colonization frequency than and Verticillium (Table 3). The presence of Alternaria in other parts of the plants throughout all stages of plant every variety and its dominance in the three tissue types development. The increased frequency may be related to examined confirm it as the dominant fungus. However, the large surface area exposed to the outer environment some genera were harbored in specific tissues; for example, and the presence of stomata providing natural ease of entry Colletotrichum, Pestalotiopsis, Stemphylium solani, and Magnaporthe to airborne and water-dispersed spores [16, 19]. The oryzae were exclusively found in cotton leaves (Table 2). endophytic colonization frequency of nine resistant cotton The roots, stems, and leaves differed in density and varieties exhibited no significant difference. No obvious composition of their endophytic communities. correlation was observed between the level of resistance There is an inherent general trade-off between fast

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growth (high colonization) and production of antibiosis. cotton-endophyte interactions and distribution. They also Endophyte species common in the host plants under provide a platform for screening novel endophytes with natural conditions are often good colonizers and grow fast broad-spectrum resistance to cotton diseases and the in vitro. On the other hand, antibiosis producers usually purification of novel natural antimicrobial agents from appear to be relatively rare in nature, tend to grow slowly G. hirsutum endophytic fungi. Combined greenhouse and in vitro, and usually are not good colonizers [36]. The field studies are required to evaluate the usefulness of endophyte Leptosphaeria sp., which was only isolated once, these endophytic biocontrol agents. grew slowly in vitro but exhibited the highest inhibition activity against V. dahliae Vd080 in the dual culture Acknowledgments antagonistic bioassay. It produced multiple volatile and nonvolatile antifungal substances. The Leptosphaeria sp. The authors wish to thank the National Science Foundation (CEF-714) nonvolatiles completely restricted V. dahliae mycelial of China (No. 31201466) and the National High-tech R and growth (Table 7 and Fig. 2). Eight isolates, belonging to the D Program of China (863 Program) (No. 2013AA102601) for most encountered fast-growing group Alternaria, exhibited the financial support. variable V. dahliae inhibitory abilities. The metabolites secreted by A. brassicae did not affect normal V. dahliae References growth. A. tenuissima inhibitory activity was moderate, and the remaining Alternaria species exhibited low or slight 1. Abdalla MH, El-Tayeb NM. 1981. Preliminary survey of antifungal activity against the target pathogen (Table 7). cotton flower mycoflora from Sudan. Trans. Br. Mycol. Soc. It is clear that the in vitro results do not necessarily 76: 367-370. directly reflect what occurs in plants; however, they are 2. Al-Samarrai TH, Schmid J. 2000. 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