Diversity of Endophytic Fungi from Different Verticillium-Wilt-Resistant Gossypium hirsutum and Evaluation of Antifungal Activity Against Verticillium dahliae In Vitro
Diversity of Endophytic Fungi from Different Verticillium-Wilt-Resistant Gossypium hirsutum and Evaluation of Antifungal Activity Against Verticillium dahliae In Vitro
Journal of Microbiology and Biotechnology. 2014. Sep, 24(9): 1149-1161
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : February 18, 2014
  • Accepted : May 16, 2014
  • Published : September 28, 2014
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About the Authors
Zhi-Fang, Li
Ling-Fei, Wang
Zi-Li, Feng
Li-Hong, Zhao
Yong-Qiang, Shi
He-Qin, Zhu

Cotton plants were sampled and ranked according to their resistance to Verticillium wilt. In 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. However, no significant difference in the abundance of isolated endophytes was found among resistant cotton varieties. Alternaria exhibited the highest colonization frequency (7.9%), followed by Acremonium (6.6%) and Penicillium (4.8%). Unlike tolerant varieties, resistant and susceptible ones had similar endophytic fungal population compositions. In three Verticillium-wilt-resistant cotton varieties, fungal endophytes from the genus Alternaria were most frequently isolated, followed by Gibberella and Penicillium . The maximum concentration of dominant endophytic fungi was observed in leaf tissues (0.1797). The evenness of stem 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 and provide a platform for screening G. hirsutum endophytes with biocontrol potential.
Endophytes are microorganisms that inhabit healthy plant tissues without causing any apparent or detectable symptoms in the host [39] . The topographical term was expanded to include fungi, bacteria, and actinomycetes, which spend either the whole or a period of their life cycle colonizing the symplastic or apoplastic spaces of asymptomatic living plant tissues [55] . However, the definition of an endophyte has been broadened and can include any organism that lives in plant tissue whether neutral, beneficial, or detrimental [43] . Based on their nature, endophytes can be divided into three categories: (i) nonpathogenic to own host but are pathogens of other hosts; (ii) nonpathogenic microbes; and (iii) pathogens that have been rendered nonpathogenic but are still capable of colonization by selection methods or genetic alteration [5] . In most cases, the relationship between an endophyte and the host is neither pathogenic nor simply mutualistic [51 , 55] ; however, host plants sometimes benefit from coexisting endophytes that provide resistance to biotic and abiotic stresses [19 , 27] . Specifically, fungal endophytes can directly inhibit pathogen infection and proliferation within the host via antibiosis, competition, and mycoparasitism [6 , 36] , or indirectly by inducing resistance responses intrinsic to the host against the fungal pathogens [7] .
Cotton is one of the most important natural fiber crop plants worldwide, with a high economic value. Verticillium wilt disease, caused by the phytopathogenic fungus Verticillium dahliae Kleb., is the major threat to cotton production [24 , 56] . V. dahliae hyphae grow from the root surface toward the cortical tissue, which is adjacent to the stele, and subsequently attack the aerial parts of the plant, resulting in systematic disease [11] . This vascular disease of cotton cannot be prevented using cultivars or cultural practices alone [42] . Moreover, traditional methods of chemically controlling these diseases can be expensive and ineffective, and have a negative impact on both environmental and human health. Biological control as part of integrated pest management has been suggested as the most sustainable long-term solution [10] . As a new source of biological control agents, the use of cotton fungal endophytes for combating Verticillium wilt disease requires further investigation.
Previous studies have reported on the diversity of the mycoflora community in flowers, the incidence of Fusarium species associated with roots, and the recovery frequency of fungal endophytes isolated from the stems of native Gossypium species [1 , 51 , 57] . The endophytic fungal diversity of transgenic (expresses the Cry1Ac protein) and isoline cotton exhibits consistent patterns in the leaves, stems, and roots. However, the tissue type and developmental stage significantly affect the fungal community composition [50] . A few cotton endophytes have been identified as plant-growth-promoting and biocontrol agents. Cladorrhinum foecundissimum , an endophytic fungus obtained from cotton stem tissue, colonizes the root tissues and accelerates the level of phosphorus uptake and plant growth [18] . The cotton endophytic fungus Lecanicillium lecanii readily colonized two potential hosts and was transferred between aphids and cotton plants, killing aphids but not causing obvious disease in cotton leaves [3] . According to Wang et al . [51] , none of the endophytic fungal isolates recovered from cotton induced foliar symptoms by either root dipping or stem puncturing inoculation. This finding supports the theory that Gossypium endophytic fungi are unlikely sources of cotton pathogens.
Although there are a few fragmentary reports on G. hirsutum fungal endophytes, no comprehensive work has been carried out on either their distribution in tissues or variability in resistance. Far less work has been conducted on the evaluation of their biocontrol potential. The objectives of this study were (i) to isolate and identify the fungi that inhabit the healthy internal tissues from cotton varieties of varying resistance to Verticillium wilt; (ii) to clarify the distribution patterns of endophytic fungi in different tissues and cotton varieties and calculate the colonization frequency and diversity indices; and (iii) to screen endophytic fungi with high-level inhibitory activity and evaluate their potential as V. dahliae biocontrol agents. This work is the first systematic report on the incidence of endophytic fungi from G. hirsutum and their activity against the destructive cotton pathogen V. dahliae .
Materials and Methods
- Collection of Cotton Varieties
Twelve varieties ranked in accordance to Verticillium wilt resistance were selected. Jimian 11, Zhongmiansuo 8, and Zhongmiansuo 24 are V. dahliae susceptible, whereas Zhongzhimian 2, Glucanase-Chitinase transgenic cotton, and Haidaomian are resistant. The other six varieties formed the tolerant group ( Table 1 ). All of the cotton plants were cultivated in Anyang, Henan Province, China. At the flowering stage, five healthy plants of each variety were collected and separated into leaves, stems, and roots.
The number of endophytic fungi isolated from the tissues of variably resistant cotton varieties.
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aV. dahliae resistance varied among the cotton varieties. S, T, and R indicate susceptible, tolerant, and resistant, respectively.
- Source of V. dahliae Strain
The strongly virulent defoliating V. dahliae isolate Vd080 recovered from Verticillium-wilt-symptomatic cotton in Hebei Province, China was used for the antagonism test. The isolate was single-spore purified for three generations and stored at -70℃ in 15% glycerol for long-term preservation and kept at 4℃ for 2 days before use.
- Surface Sterilization and Isolation of Endophytic Fungi
The root, stem, and leaf segments were washed in flowing tap water to remove epiphytes and soil debris. The stem and root tissues were then rinsed in 75% ethanol for 3 min, and in 5% NaClO for 1 min, and then washed three times with sterile water. The leaves were rinsed for 2 min in 75% ethanol, followed by 30 sec of 5%-NaClO and sterile water washing.
After surface drying, each segment was cut into 0.5 × 0.5 cm 2 pieces with a sterile blade. Fifty pieces of each tissue from the different cotton varieties were placed onto PDA plates (five pieces in each plate) amended with 200 ml/l streptomycin (to prevent bacterial contamination). To validate the effects of surface sterilization, the suspension collected from the last rinsing step was plated onto PDA medium. To test the sterility of the bench, an uncovered blank PDA plate was placed on it for 30 min and then incubated at 25℃ for 2 days. The tissues were observed for fungal growth on alternate days for 15 days. Actively growing fungal tips emerging from plant tissues were subcultured on PDA for identification and enumeration.
All of the endophytic fungi isolated were assigned specific code numbers (CEF-001-CEF-842) and maintained on PDA slants at 4℃. All of the samples were deposited at the Plant Protection Department, Institute of Cotton Research, CAAS.
- Molecular Identification of Endophytic Fungi
Pure endophytic fungi were cultured with agitation in Czapek Dox liquid medium (NaNO 3 , 2.00 g; K 2 HPO 4 , 1.00 g; KCI, 0.50 g; MgSO 4 ·7H 2 O, 0.50 g; FeSO 4 , 0.01 g; sucrose, 30.00 g; H 2 O, 1,000 ml) at 25 ± 1℃ for 7 days. Genomic DNA was extracted from frozen mycelia following the method described by Al-Samarrai and Schmid [2] . PCR amplification of the internal transcribed spacer (ITS) region of the fungal DNA was conducted with the following universal fungus-specific primers: ITS1 (5’-TCCGTAGGTGAA CCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) [54] . PCR amplifications were performed with a Dongsheng EDC-810 thermocycler (Dongsheng Innovation Biotech Co., Ltd., Guangdong, China) in a total reaction volume of 50 μl, containing 1× Taq buffer, 0.5 U of Taq DNA polymerase (Takara, Dalian, China), 2 mM dNTPs, 2 μM of each primer, and approximately 25 ng of template DNA. The initial DNA denaturation step was performed at 94℃ for 2 min, followed by 30 cycles of denaturation at 94℃ for 1 min, annealing at 55℃ for 30 sec, and extension at 72℃ for 1 min, with a final extension at 72℃ for 10 min. ITS products were sequenced by the GENEWIZ Corporation, Suzhou, China. The resulting sequence of the ITS rDNA was compared with those available in GenBank using the BLAST network service to determine their phylogenetic affiliation. The phylogenetic analysis was performed using MEGA ver. 5.05 after multiple alignment of the data by Clustal_X [46] , with gaps treated as missing data. Clustering was performed using the neighbor-joining method [41] . Bootstrap analysis was used to evaluate the tree topology of the neighbor-joining data by performing 1,000 resamplings [14] . The ITS rDNA sequence was deposited in the GenBank nucleotide sequence database under accession numbers KF998977-KF999032 and KF657726-KF657729.
- Statistical Analysis
The colonization frequency (% CF) of fungal species was calculated according to Hata and Futai [23] as follows:
CF = (N col /N t ) × 100, where N col = number of segments colonized by each fungus, and N t = total number of segments examined.
The isolation recovery (IR) of a single endophytic taxon from the cotton tissue was calculated according to the following formula:
IR = Ni/Na × 100, where Ni = number of each fungus recovered from the given tissue, and Na = total number of fungi isolated from the given tissue.
The fungal endophytic diversity of each cotton variety and the different cotton tissues was estimated with five diversity indices. The reason for using these diversity indices was to take advantage of the strengths of each index and to predict the complete structure of different populations. Simpson’s index of dominance (D) was calculated by the following formula [44] :
D = Σ(n/N) 2 , where n = the total number of isolates of a particular species, and N = the total number of isolates of all species.
Simpson’s diversity index = 1 − D.
Species richness = S /
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, where S = total number of species.
Shannon-Wiener index (H’) = −Σ(piInpi), where pi = n/N.
Species evenness E = H’/InS.
- In Vitro Fungal Endophytic Antagonism Test
The antifungal activity of isolated endophytic fungi against the V. dahliae Vd080 isolate was evaluated by three different methods. These were dual culture and volatile and nonvolatile metabolite bioassays. Each treatment had three replicates and each experiment was conducted twice.
- Dual Culture Antagonistic Bioassay
The dual culture technique was used to assay the antagonistic activity of endophytic fungal isolates. A plug of 5 mm 2 diameter 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 25 ± 1℃. The plate was then inoculated with a 5-mm 2 -diameter mycelial disc of the endophytic isolate, placed 5 cm from the pathogen at the opposite side, and incubated at 25 ± 1℃. A plate inoculated with V. dahliae alone served as a control. Each treatment had three replicates. The growth inhibition rate was calculated.
- Volatile Metabolite Inhibitory Bioassay
The effect of volatile antibiosis of endophytic fungi on Vd080 was tested in the assemblage described by Dennis and Webster [12] and Naraghi et al . [38] . A 5 mm 2 disc of Vd080 was placed on petri dishes containing PDA medium and incubated at 25 ± 1℃ for 4 days. Then, 5 mm 2 discs of endophytic fungi were also cultured on petri dishes containing PDA medium. Two petri dish bottoms (without their lids) containing pathogen and antagonist isolates were placed face to face and then sealed with thin plastic film. The control petri dishes were not inoculated with antagonistic fungi. The petri dishes were incubated at 25 ± 1℃.
- Nonvolatile Antibiotic Inhibitory Assay
The effect of accumulation of nonvolatile antibiotics produced by endophytic fungi on Vd080 growth was examined using the exudate filtering method [53] . Petri dishes containing PDA medium were completely covered with two layers of autoclaved cellophane and centrally inoculated with 5 mm 2 discs of endophytic fungus. The plates were incubated for 5–7 days at 25 ± 1℃ before the cellophane and antagonist were removed. A 5 mm 2 pathogen disc was then placed in the center of the dish. The dishes were incubated for a further 15 days before radial growth was recorded and compared with that of the control plates.
- Growth Inhibition Analysis
The Vd080 mycelial colony growth (mm) data was recorded at 2-day intervals. A final observation on radial mycelial colony growth was made after approximately 7–15 days, when the presence of overlapping fungi on any one petri dish was recorded. The growth inhibition rate (GI) was calculated using the following formula given by Whipps [52] :
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where R 1 is the radius length of the V. dahliae isolate Vd080 as control. For the dual culture antagonistic bioassay, R 2 is the radial distance of Vd080 grown on a line between the inoculation of the pathogen and endophytic fungus. For the volatile and nonvolatile metabolite bioassays, R 2 is the radial distance of Vd080 grown with endophytic fungi. An inhibitory effect of more than 75% was considered strong inhibition, 50–75% moderate, 25–50% low, and 10–25% slight.
- Isolation of Endophytic Fungi from Cotton
Endophytic fungi were isolated from asymptomatic root, stem, and leaf segments of cotton plants following a standard isolation protocol. In total, 642 endophytic fungi were obtained from 12 Verticillium-wilt-resistant cotton varieties, consisting of three susceptible, three resistant, and six tolerant varieties. The healthy living tissues of susceptible cotton plants harbored 176 endophytic fungi with an average of 59 per variety. In the three resistant varieties, 171 fungi were observed in the internal tissues with an average of 57 per variety. In the tolerant varieties, 295 endophytic fungi were isolated from six plants with an average number of 49 per variety. Of the 12 cotton varieties, the susceptible cotton JM11 had the largest population ( n = 96). However, ZM8, which was also a susceptible variety, carried the least endophytic fungi ( n = 29). The abundance of isolated endophytic fungi did not differ significantly among the resistant cotton varieties ( Table 1 ).
The populations of endophytic fungi associated with different cotton tissue types were also compared. The distribution of endophytic fungi within cotton was not uniform. With the exception of JM668, a tolerant variety, the number of fungi found in the leaves was greater than in both the roots and stems. Out of 642 endophytic fungi isolates, the majority ( n = 391, 60.9%) were found in the leaves, followed by the roots ( n = 140, 21.8%), and stems ( n = 111, 17.3%) ( Table 1 ).
- Identification and Distribution of Endophytic Fungi in Different Tissues
Based on the ITS phylogenetic analysis, 642 endophytic fungi were divided into 27 groups representing 27 different genera, including 60 species. With the exception of Rhizoctonia solani , which belongs to the phylum Basidiomycota, all of the isolates were members of the phylum Ascomycota. This included members of the classes Sordariomycetes, Dothideomycetes, Eurotiomycetes, and Saccharomycetes ( Fig. 1 ).
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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.
Alternaria , a Dothideomycete, exhibited the highest total colonization frequency (CF = 7.9%). Out of 642 endophytic fungal isolates, 143 belonged to this group, which comprised eight species. Of these, Alternaria alternata was the most common. The second largest endophyte genus was Acremonium , which comprised four species and contained 119 isolates with a CF of 6.6%. Penicillium , which represented the third most dominant group, comprised 87 endophytic fungi and had a CF value of 4.8% ( Table 2 ).
The distribution and frequency of endophytic fungi isolated from cotton tissues.
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*At least two species of the genus were found. aThe number of isolates; bisolation recovery; ccolonization frequency.
The endophytic fungi harbored in the cotton roots, stems, and leaves exhibited a diverse population structure. In root tissues, 140 fungal isolates belonging to 19 genera were recovered from 600 segments. Of these, Gibberella exhibited the highest colonization frequency (5.2%) and isolation recovery (22.5%), followed by Penicillium (CF = 4.8%, IR = 20.7%) and Acremonium (CF = 4.0%, IR = 17.1%). Leptosphaeria , Trichoderma atroviride , Ascomycete , and Phoma were only isolated from root tissues in this experiment. In stem tissues, 19 genera were identified in all. The most common genus was Gibberella with the highest colonization frequency (4.0%) and isolation recovery (21.7%), followed by Penicillium and Acremonium , Rhizoctonia solani , Plectosphaerella , and Meyerozyma caribbica , which only appeared in stem tissues. The group Alternaria was the largest population in the fungal endophytes isolated from cotton leaves (CF = 20.7%, IR = 31.7%). The second most dominant species was Trichoderma atroviride with 13.3% CF and 20.5% IR. Colletotrichum , Pestalotiopsis , Stemphylium solani , and Magnaporthe oryzae were found exclusively in cotton leaves ( Table 2 ).
The most common species isolated from the roots, stems, and leaves were Alternaria , Penicillium , Pichia guilliermondii , Acremonium , Aspergillus , Trichothecium roseum , Bionectria , Chaetomium globosum , Cladosporium , Fusarium , Talaromyces , Gibberella , and Verticillium . The 13 common taxa, except for Verticillium and Chaetomium globosum , isolated from the leaves had a slightly lower colonization frequency than those from the roots and stems. The other 12 genera or species recovered from the leaves exhibited higher colonization frequencies ( Table 3 ).
Colonization frequency (%) of endophytic fungi common to all three tissue types.
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Colonization frequency (%) of endophytic fungi common to all three tissue types.
- Incidence of Fungal Endophytes in Verticillium-Wilt-Resistant Cotton Varieties
The population composition of endophytic fungi varied among susceptible, resistant, and tolerant cotton plants. A total of 176 isolates, representing 19 genera, were isolated from three susceptible cotton varieties, which were JM11(96), ZM8(29), and ZM24(51). In JM11, 58 Alternaria species accounted for 60.4% of the total isolates. However, in the ZM8 family, the dominant group was Gibberella , accounting for 31%. The endophytic fungi originating from ZM24 exhibited a different population structure. The biggest group, Acremonium , had 17 isolates, accounting for 33.3%.
The endophytic fungal population composition of resistant 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 widespread occurrence of the genus Alternaria , only one Alternaria sp. was isolated from ZZM2. Gibberella was the most common genus in the transgenic cotton Glu-Chi, and Alternaria represented the main group of endophytic fungi recovered from HDM. In three Verticillium-wilt-resistant cotton varieties, fungal endophytes of the genus Alternaria were isolated most frequently, followed by Gibberella and Penicillium .
Compared with the susceptible and resistant cotton varieties, there were some notable differences in the representation of some genera across the six tolerant varieties. Acremonium was the most dominant group, with the most isolates being recovered from ZM35, YM2067, and LM28. The genus Gibberella represented the largest endophytic fungal population from JM668. In LM21 and the PCD transgenic cotton varieties, Fusarium was the largest proportion of the endophytic fungal population ( Table 4 ).
The distribution of endophytic fungi in cotton varieties of varying resistance toV. dahliae.
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The distribution of endophytic fungi in cotton varieties of varying resistance to V. dahliae.
- Endophytic Community Diversity Analysis
The diversity of the endophytic communities isolated from different tissues and cotton varieties was compared using indices of α-diversity (Simpson's diversity index and Shannon-Wiener index) and their components, including Simpson’s dominance, and species richness and evenness.
The concentration of dominance or Simpson’s dominance of endophytic fungi was highest in the leaf tissues (0.1797). A large index value proportionally reflects greater species diversity. That is, leaf endophytes were more diverse than those of the other two tissues. Both Simpson’s and Shannon-Wiener diversity indices (0.8692, 2.3393) were highest in stem tissue endophyte communities. Species richness was also greatest in stem endophytes (2.6576). The evenness of all communities was also calculated in which the stem tissue endophytic community (0.702) was comparatively more uniform than the other two tissues ( Table 5 ).
Diversity indices of endophytic fungi colonizing cotton root, stem, and leaf tissues.
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Diversity indices of endophytic fungi colonizing cotton root, stem, and leaf tissues.
Endophytic fungi from the tolerant cotton PCD had the highest Simpson’s (0.8791) and Shannon-Wiener diversity indices (2.2045); the lowest endophyte diversity was from susceptible cotton variety JM11. The ZM8 endophytic community had the greatest species richness (5.1995), followed by ZM24 and LM21. The numbers of individual species were most evenly distributed in the PCD endophytic community (0.6616), followed by ZZM2 and HDM ( Table 6 ).
Diversity indices of endophytic fungi from different cotton varieties.
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Diversity indices of endophytic fungi from different cotton varieties.
- V. dahliae Inhibitory Activity of Endophytic Fungi
A total of 80 endophytic fungi selected from 27 genera were evaluated for their V. dahliae inhibitory activity in vitro . The results of the dual culture antagonistic bioassay showed that endophytes isolated from cotton varied greatly in their fungistatic activities against this cotton pathogen. Nearly half of the endophytes (41 isolates) tested had no effect on normal V. dahliae growth; the antibiotic ability of the remaining 39 isolates was analyzed further. Out of 39, seven (17.9%) endophytic fungi exhibited strong antifungal activity toward Vd080 with high growth inhibition rates (≥75%). CEF-714, which was identified as Leptosphaeria sp., exhibited maximal inhibition ability with the mean growth inhibition rate being 88.1%. V. dahliae growth was also strongly inhibited by CEF-642, Talaromyces flavus (GI = 83.8%). In addition, the isolates CEF-026 ( Gibberella intermedia ), CEF-193 ( Acremonium sp.), CEF-325 ( Fusarium solani ), CEF-818 ( Penicillium simplicissimum ), and CEF-109 ( Ascomycete sp.) belonged to this strongly antagonistic group. CEF-421 ( Aspergillus aculeatus ), CEF-718 ( Penicillium sp.), CEF-106 ( Talaromyces stollii ), and CEF-070 ( Alternaria tenuissima ) comprised a moderately antifungal group with growth inhibition rates ranging from 50% to 75%. The other 28 isolates, including 17 with low and 11 with slight growth inhibition levels, exhibited reduced fungistatic activity ( Table 7 ). The antagonistic group that comprised seven endophytic cotton fungi was selected to test both volatile and nonvolatile V. dahliae fungistasis.
The dual cultural antagonistic activity of 39 endophytic fungal isolates againstV. dahliae.
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aGrowth inhibition rate.
CEF-193 volatile substances exhibited the most V. dahliae inhibition activity, having a growth inhibition rate of 94.3%. CEF-642 also strongly reduced the growth of the targeted pathogen, with an 81.6% inhibition rate. CEF-818, CEF-714, and CEF-325 also produced volatile metabolites that greatly inhibited Vd080 growth ( Fig. 2 ).
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The volatile antagonistic activity of cotton endophytic fungi against V. dahliae.
The results of the nonvolatile antibiotic assay revealed that CEF-818, CEF-325, CEF-714, and CEF-642 secreted the highest amounts of V. dahliae resistant nonvolatile substances. The V. dahliae mycelia did not spread normally on the PDA plates containing the antibiotic substances mentioned above. The inhibition rate reached to 100%. V. dahliae growth was moderately suppressed by CEF-193 nonvolatiles. However, the interaction between CEF-026 nonvolatiles and Vd080 was slight. The inhibition rate was only 13.7% ( Fig. 3 ).
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The nonvolatile antagonistic activity of cotton endophytic fungi against V. dahliae.
Endophytes inhabit healthy plant tissues, which provide nutrition and shelter. At the same time, they are viewed as an excellent source of bioactive compounds for the host [31 , 49] . They also produce many functional metabolites that impact plant health and growth [37] . Of these, some signal metabolites independently trigger plant defense cascades as pathogens of other hosts (systemic acquired resistance) and nonpathogens (induction of systemic resistance), providing higher levels of pathogen resistance in plants [5 , 40] .
It is clear that conducting a comprehensive study of any host plant is necessary before screening the potential of their endophytic fungi. In this study, 642 endophytic fungi were obtained from the roots, stems, and leaves of 12 cotton varieties with varying resistance to Verticillium wilt. In accordance with previous reports [19 , 25 , 32 , 50] , the number of endophytes recovered from the leaves (391) was significantly larger than that from roots and stems. Most leaf endophytes had a greater colonization frequency than other parts of the plants throughout all stages of plant development. The increased frequency may be related to the large surface area exposed to the outer environment and the presence of stomata providing natural ease of entry to airborne and water-dispersed spores [16 , 19] . The endophytic colonization frequency of nine resistant cotton varieties exhibited no significant difference. No obvious correlation was observed between the level of resistance 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 not be identified, which is a problem commonly reported in the literature [4 , 17 , 22 , 25 , 30] . Molecular methods have greatly increased our knowledge regarding fungal diversity and taxonomy. Several recent studies have shown that molecular methods can be successfully used in the study of endophytic fungi [35 , 47] . The most widely used gene regions for the detection of endophytic fungi are the ribosomal DNA sequences, especially the internal transcribed spacer region (ITS1, 5.8S, and ITS2) [9 , 34] . Based on the ITS characteristics, 642 endophytic cotton fungi were successfully identified, representing 27 different genera and 60 species.
The G. hirsutum endophytic assemblage is composed of a number of cosmopolitan genera such as Alternaria , Acremonium , Penicillium , Gibberella , and Fusarium . The former three genera were the most frequently isolated from G. hirsutum . The dominant group was Alternaria with 143 members comprising eight species. This finding is in accordance with a previous report on the endophyte frequency of four native Gossypium species in Australia [51] . In addition, Alternaria , Gibberella , and Fusarium have been reported as the most encountered endophytes in various types of plants [20 , 29] . Thirteen common taxa in this study were Alternaria , Penicillium , Pichia guilliermondii , Acremonium , Aspergillus , Trichothecium roseum , Bionectria , Chaetomium globosum , Cladosporium , Fusarium , Talaromyces , Gibberella , and Verticillium ( Table 3 ). The presence of Alternaria in every variety and its dominance in the three tissue types examined confirm it as the dominant fungus. However, some genera were harbored in specific tissues; for example, Colletotrichum , Pestalotiopsis , Stemphylium solani , and Magnaporthe oryzae were exclusively found in cotton leaves ( Table 2 ). The roots, stems, and leaves differed in density and composition of their endophytic communities.
There is an inherent general trade-off between fast growth (high colonization) and production of antibiosis. Endophyte species common in the host plants under natural conditions are often good colonizers and grow fast in vitro . On the other hand, antibiosis producers usually appear to be relatively rare in nature, tend to grow slowly in vitro , and usually are not good colonizers [36] . The endophyte Leptosphaeria sp., which was only isolated once, grew slowly in vitro but exhibited the highest inhibition activity against V. dahliae Vd080 in the dual culture antagonistic bioassay. It produced multiple volatile and nonvolatile antifungal substances. The Leptosphaeria sp. (CEF-714) nonvolatiles completely restricted V. dahliae mycelial growth ( Table 7 and Fig. 2 ). Eight isolates, belonging to the most encountered fast-growing group Alternaria , exhibited variable V. dahliae inhibitory abilities. The metabolites secreted by A. brassicae did not affect normal V. dahliae growth. A. tenuissima inhibitory activity was moderate, and the remaining Alternaria species exhibited low or slight antifungal activity against the target pathogen ( Table 7 ).
It is clear that the in vitro results do not necessarily directly reflect what occurs in plants; however, they are particularly useful for identifying candidates with biocontrol potential to reduce pathogen damage. In this study, seven endophytic fungi with strong V. dahliae antifungal activity were identified as candidates for the biocontrol of this cotton disease ( Table 7 , Figs. 1 and 2 ). Of these, Talaromyces flavus has been studied previously. T. flavus antagonizes V. dahliae by parasitism and antibiosis [13] . V. dahliae microsclerotia are killed by T. flavus culture filtrate, the toxicity of which has been attributed to the action of glucose oxidase, chitinase, and cellulose [15 , 28] . In addition, the endophytic fungus Fusarium sp. from the plant Selaginella pallescens was screened for antifungal activity, and a new pentaketide antifungal agent (CR377) was isolated [8] . Penicillium reduces both Fusarium and Verticillium wilt in tomatoes by inducing resistance in plants; this group also stands out as having high biocontrol potential [33] .
Fungal endophytes are well known for their ability to produce a wide range of antimicrobial substances and enhance plant resistance to pathogens and pests [26 , 45] . Microbial metabolites may have an active role in disease resistance development by functioning as signals mediating cross-talk between the endophytes and their hosts [21 , 40] . Therefore, this study describes a comprehensive endophytic assemblage inhabiting G. hirsutum and highlights the potential of selected microbes against the main cotton pathogen V. dahliae .
These findings further add to the understanding of cotton-endophyte interactions and distribution. They also provide a platform for screening novel endophytes with broad-spectrum resistance to cotton diseases and the purification of novel natural antimicrobial agents from G. hirsutum endophytic fungi. Combined greenhouse and field studies are required to evaluate the usefulness of these endophytic biocontrol agents.
The authors wish to thank the National Science Foundation of China (No. 31201466) and the National High-tech R and D Program of China (863 Program) (No. 2013AA102601) for the financial support.
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