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Biocontrol potential of endophytic fungi in medicinal plants from Wuhan Botanical Garden in China
Accepted Manuscript Biocontrol potential of endophytic fungi in medicinal plants from Wuhan Botanical Garden in China Libo Xiang, Shuangjun Gong, Lijun Yang, Jianjun Hao, MinFeng Xue, FanSong Zeng, XueJiang Zhang, WenQi Shi, Hua Wang, Dazhao Yu PII: DOI: Reference:
S1049-9644(15)30058-X http://dx.doi.org/10.1016/j.biocontrol.2015.12.002 YBCON 3356
To appear in:
Biological Control
Received Date: Revised Date: Accepted Date:
19 August 2015 25 November 2015 4 December 2015
Please cite this article as: Xiang, L., Gong, S., Yang, L., Hao, J., Xue, M., Zeng, F., Zhang, X., Shi, W., Wang, H., Yu, D., Biocontrol potential of endophytic fungi in medicinal plants from Wuhan Botanical Garden in China, Biological Control (2015), doi: http://dx.doi.org/10.1016/j.biocontrol.2015.12.002
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Biocontrol potential of endophytic fungi in medicinal plants from Wuhan Botanical Garden in China Libo Xianga, Shuangjun Gonga, Lijun Yanga, Jianjun Haob, MinFeng Xuea, FanSong Zenga, XueJiang Zhanga, WenQi Shia, Hua Wanga ,Dazhao Yua a
Institute of Plant Protection and Soil Science, Hubei Academy of Agricultural Sciences, Key
Laboratory of Integrated Pest Management on Crop in Central China, Ministry of Agriculture, Hubei Key Laboratory of Crop Diseases, Insect Pests and Weeds Control, Wuhan 430064, China; b
School of Food and Agriculture, The University of Maine, Orono, ME 04469, USA
These authors contributed equally to this work. Corresponding author. E-mail address: [email protected],
Abstract Many medicinal plants possess antimicrobial activities, and have antagonistic endophytic fungi that help them protect from pathogen attack. The aim of this study was to examine endophytic fungi in traditional Chinese medicinal plants, and understand if these organisms have antimicrobial activities and they can be potentially used for biological control of plant diseases. A total of 208 endophytic fungal isolates were collected from stems (83), leaves (121) and flowers (4) of 26 medicinal plant species. The majority of the isolates belonged to Alternaria, Phomopsis, Colletotrichum, Phoma and Acremonium as well as several species allocated to mycelia sterilia. A detached leaf assay was conducted by testing these isolates on wheat powdery mildew (Blumeria graminis f.sp. tritici, or Bgt). Fifteen isolates of endophytic fungi inhibited Bgt, exhibiting control efficacies ranging from 65.4 to 100%. Of these isolates, LPS-1, SCS-6 and 16-6, exhibited significant inhibition of Bgt proliferation (>90%). Isolate LPS-1 isolated from the stem of Ilex cornuta Lindl. ex Paxt. had the highest efficacy, resulting in 100% inhibition of Bgt growth on detached leaf segments. Based on morphological characteristics and phylogenetic analyses of the ITS rDNA sequences and translation elongation factor 1-alpha (TEF-1α)
gene regions,
LPS-1 was
identified as Lasiodiplodia pseudotheobromae. A range of culture conditions for LPS-1 were examined and the results indicated that optimal antifungal activity resulted from static cultures in PDB (pH 7.0) inoculated with three mycelial plugs and incubated at 30°C for 6 days. With further study, LPS-1 can be a candidate for biological control. Keywords: Medicinal plants; Endophytic fungi; Lasiodiplodia pseudotheobromae; Biological control
1 Introduction Endophytic fungi play an important role in enhancing plant health, and have been recognized as an important resource of biocontrol agents to suppress plant pests including both insects and pathogens (Backman and Sikora, 2008; Kumar and Kaushik ,2013;Zhang et al.,2014). Medicinal plants could be a valuable repository for fungal endophytes yielding novel metabolites of agricultural and pharmaceutical importance (Kusari and Spiteller, 2012; Winter et al., 2011; Ji et al., 2009). In Brazil, for example, medicinal plant Baccharis trimera (Asteraceae)
harbores endophytic fungi, belonging to 25 different taxa (Vieira et al., 2014),
and exhibited a broad spectrum of inhibitory activity against a range of pathogenic bacteria. Furthermore, endophytes synthesizing bioactive metabolites can be used directly or indirectly as biocontrol agents against plant diseases (Strobel et al., 2004; Staniek et al., 2008; Aly et al., 2010; Kharwar et al., 2011; Kusari and Spiteller, 2012). One endophytic isolate of Cochliobolus sp. obtained from the plant Piptadenia adiantoides (Fabaceae) produces antifungal compounds cochlioquinone A and isocochlioquinone A (Campos et al., 2008). As a consequence the study of endophytic fungi has received more and more attention. Powdery mildew (Blumeria graminis Speer) is a severe disease, causing significant economic losses in a wide range of field crops. In China, the disease affected up to 12 million hectares in 1990 and resulted in estimated grain yield losses of 14.4 million metric tons (Liu and Shao 1994). Over the last decade, an average of more than six million hectares of growing area has been affected annually (http://www.agri.gov.cn/). To date management of this disease has largely been reliant on the application of chemical fungicides such as sterol demethylation inhibitors (DMIs). However, the development of pathogen strains resistant to
the mostly used triadimefon fungicide has been reported and the resistant strain is now widespread (Yang et al., 2011; Gong et al., 2013).
Alternatively, finding strategies such as
biocontrol for the management of this devastating disease are urgently required. Many reports have been found to be effective as biocontrol agents against various powdery mildew fungi (Elad et al., 1996; Kiss, 2003; Gao et al., 2015), but little research has been conducted on control of wheat powdery mildew using BCAs. The objectives of this study were to explore the diversity of endophytic fungi associated with traditional Chinese medicinal plants, to screen the isolates obtained for antagonistic activity with the potential to control Bgt, and third to optimize the culture conditions of candidate biocontrol agents to maximize their antifungal activity. 2. Materials and methods 2.1. Isolation of endophytic fungi Tissues of stems, leaves and flowers were collected from healthy medicinal plants at the Wuhan Botanical Garden, Chinese Academy of Sciences. The leaflets, stem and flower segments were surface sterilized according to the protocol of a previous study (Hallmann et al., 2006). The plant material was thoroughly washed using distilled water, followed by treatment with 70% ethanol for 2 min and 5% sodium hypochlorite for 5 min and then rinsed in sterile distilled water. The water from the final wash was collected and plated onto PDA as a positive control to confirm that the surface sterilization had been successful. The sterilized samples were then cut into 5 to 6 pieces (2 to 6 mm in diameter) and plated onto water agar (distilled water, 1.5% agar) amended with 100 μl/ml ampicillin. The samples were then incubated at 25°C for 3 to 14 days until fungal growth was observed on their surfaces (Hijri
et al., 2002). The emerging hyphal tips were then picked and purified by successive subculture on potato dextrose agar (PDA) plates before being maintained as stock cultures at 4°C. 2.2. Identification and diversity of endophytic fungi The isolates of endophytic fungi were provisionally identified under a microscope based on the morphology of their colonies (color and mycelial characteristics) and spores (conidia, blastospores, sporangiospores or ascospores). They were confirmed by analyzing the sequence of the internal transcribed spacer (ITS) region of their rDNA (ITS1-5.8S rDNA-ITS2),
which
were
amplified
using
the
universal
primers
ITS1
(5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) (White et al., 1990). The resulting PCR products were then cloned and sequenced according to the protocol of a previous study (Zhang et al., 2010). Cultures that failed to sporulate were grouped as mycelia sterilia, and divided into different morphospecies according to their culture characteristics. The colonization rate (CR) for each plant species was calculated as the total number of plant tissue pieces colonized by endophytes divided by the total number of pieces incubated for each plant sample expressed as a percentage, while the isolation rate (IR), which is a measure of endophyte richness of a given sample of plant tissue, i.e. the number of multiple colonization by different endophytes in a single tissue sample, was calculated as the number of isolates obtained divided by the total number of tissue pieces, but not expressed as a percentage (Huang et al., 2008). 2.3. Assay for antagonistic activities of endophytic fungi on detached leaf and wheat seedling
Culture filtrates from the endophyte isolates were prepared as follows: mycelial plugs from each isolate were inoculated in 100 ml potato dextrose broth (PDB: 200g/l potato and 20 g/l sucrose) in a 250 ml Erlenmeyer flask and incubated with shaking (150 rpm) at 28°C for 7 days. The resulting cultures were then centrifuged at×8000g at 4°C, filtered through 0.22 μm membrane filters (EMD Millipore Corporation, Billerica, MA, USA) to remove hyphal fragments and stored at 4°C until required. A total of 208 isolates were assessed for their antifungal activity against Bgt using a detached leave assay described in a previous study (Yang et al. 2008). Leaf segments (2.5 cm in length) were cut from wheat plants (cv. Chancellor) at the two-leaf stage and placed randomly on 9 cm petri dishes containing 0.5% benzimidazole water agar (w/v). The culture filtrates (4 mL) were then applied to the plates using a potter spray tower (model PDE0012, Burkard Scientific Ltd, Middlesex, UK). Bgt conidia were produced by inoculating detached, surface-sterilized leaves of wheat cv. Chancellor, and then placed in a growth chamber at 18±1◦C and constant light. After air-drying for 12 h, the treated plates were inoculated at a density of 2-4×103 conidia/cm2 in a setting tower and incubated in a growth chamber with a 12 h photoperiod and 70% relative humidity. Negative and positive controls were prepared for each endophyte isolate using sterile PDB media and 10 μg/ml triadimefon, respectively, which were similarly applied using the potter spray. The disease severity was evaluated at 12 days post inoculation (dpi) using the method described in a previous study (Curtis et al., 2012) and the following scale:
0=no visible pathogen
colonization of the leaves; 1=1–5%, 2=6–10%, 3=11–25%, 4=26–50%, 5=51–75% and 6=76–100% of leaf area covered by the pathogen. Each treatment was represented by three
replicate dishes each containing ten leaf segments, and the entire experiment was conducted twice. After preliminary screening using the leaf segment assay, 15 endophytic isolates exhibiting antifungal activity were selected for further investigation using a wheat seedling assay. Fifteen wheat seeds (cv. Chancellor) were sown in pots (10 cm in diameter) containing soil amended with 0.5% (w/w) of a commercial fertilizer (N: P: K = 15:15:1; Hubei Dong Sheng Chemicals Group Co., Ltd., Yuan An County, Hubei, China). The pots were placed in plastic boxes with a shallow water level to maintain high humidity and kept in a growth chamber at 18°C with a 12 h light/12 h dark photoperiod until the emerged seedling were ten days old. Culture filtrates from the endophytic fungi were then sprayed on the seedlings, which were allowed to air dry for approximately 24 h before being inoculated with the equivalent of 2-4×103 conidia/cm2 using a miniature settling tower, consisting of a 2 L cup with a 1.5 cm opening in its top to allow aerial distribution of the test conidia onto the leafs located on the plants below. The seedlings were then placed in a growth chamber and maintained under the same conditions as described above, before the disease severity was evaluated at 12 dpi using the 0-7 scale from the detached leaf assay. The experiment had a completely randomized design, using three replicates for each endophyte isolate and negative and positive controls. The commercial fungicide triadimefon as positive control (96 % active, Jiangsu Runfeng Agrochemicals Co., Ltd., Jiangsu, China) prepared in accordance with the detached leaf assay described above. The experiments were performed twice and three replicates were used in each experiment. Data from repeated experiments were pooled before statistical analyses.
2.4. Identification of the endophytic fungus LPS-1 A preliminary identification of isolate LPS-1 was made by examining its colony characteristics, and microscopic analysis of its hyphal and conidial morphology. Sporulation was induced by culture on 2% water agar (WA) on top of which sterilized pine needles had been placed and incubation at 25°C for 5 weeks under near UV light (Ismail et al., 2012). The morphology (cell wall, shape, color, and presence or absence of septa) of the conidia within the pycnidia was assessed using a compound microscope (Nikon Inc. Instruments Group, Elville, NY). Having established that LPS-1 was likely to belong to the genus Lasiodiplodia, specifically
Lasiodiplodia
pseudotheobromae.Multigene
phylogenetic
analyses
were
conducted based on DNA sequence comparisons of the ITS rDNA sequences and translation elongation factor 1-alpha (TEF-1a) gene region (Vilgalys and Hester, 1990). Before conducting the phylogenetic analyses, DNA extraction, DNA amplification and DNA sequencing of the selected isolates were performed using the method described in zhang et al(2010). he ITS and TEF-1αsequences. The sequences of the ITS and TEF-1αgene regions were deposited in GenBank (http://www.ncbi.nlm.nih.gov) The sequences of the type specimen strains that were related to the fungus isolated in this study were obtained from GenBank. Sequence alignment and manual editing of the sequence data were performed using the Bioedit software package (http://www.mbio.ncsu.Edu/BioEdit/bioedit.html). Each of the ITS, TEF-1αand the combined ITS and TEF-1αdatasets was analyzed using the maximum-likelihood (ML) method using Molecular Evolutionary Genetics Analysis (MEGA) version 4.0 software (Tamura et al., 2007). Guignardia philoprina
(CBS446.783) was used as the out-group taxon. 2.5. Optimization of medium and culture conditions to maximize the antifungal activity of LPS-1 The optimal medium for the antifungal activity of LPS-1 as well as the effect of static or rotary culture was determined using four different media: PDB, modified potato dextrose broth (MPDB: 200 g/l potato, 20 g/l sucrose, 19.95 g/l malt extract, 0.5 g/l MgSO•7H2O, and 7.5 g/l NaNO3), malt extract broth (MEB: 20 g/l sucrose, 19.95 g/l malt extract, 0.5 g/l MgSO •7H2O, and 7.5 g/l NaNO3), and and Czapek-Dox broth (CDB: 30 g/l sucrose, 1 g/l KH2PO4, 0.5 g/l MgSO•7H2O, 3 g/l NaNO3, 0.5 g/l KCl, and 0.01 g/l FeSO4•7H2O). Liquid cultures (pH 7.0) were prepared by inoculating 100 ml medium with three mycelial plugs (5 mm in diameter) and incubating at 30°C for six days. The culture filtrates were then collected and assessed using the detached leaf assay described above (2.4). The optimal timing of antifungal activity was also evaluated using PDB liquid cultures as described above and seven different time points: 3, 4, 5, 6, 7, 8, and 9 days. Similar experiments were then conducted to assess the effect of inoculum load (3, 4, 5, 6 and 7 mycelial plugs) and pH (6.0, 6.5, 7, 7.5, and 8), both of which involved 6 days incubation at 28°C, as well as the effect of temperature (20, 25, 30, 35, and 40°C). The experiments were performed twice and three replicates were used in each experiment. Data from repeated experiments were pooled before statistical analyses. 2.6. Data analysis Data were analyzed using the SAS software package (SAS Institute, Cary, NC, USA, Version 8.0, 1999). Analysis of variance was performed, and means of treatment effects were
seperated using Duncan’s multiple range test at the significance level α=0.05. In order to compare the treatments with those applied separately, disease severity was transformed into percentages of control efficacy as follows: Efficacy = (C−T)/C] × 100, where C is the disease severity in the control (treated with PDB medium) and T is the disease severity in the examined treatment 3. Results 3.1. Diversity of endophytic fungi from Chinese medicinal plants A total of 208 endophytic fungal strains were isolated from the 26 traditional Chinese medicinal plants. A total of 40 different morphospecies were identified belonging to 17 taxonomic groups. The majority of the isolates were classified into six groups (Table 1) with the highest number being allocated to the mycelia sterilia (29.8%). The second most abundant group was the Phomopsis, which represented 18.8% of the isolates, followed by the Acremonium, Phoma, Alternaria and Colletotrichum, which had relative frequencies of 11.5%, 10.6%, 9.1% and 7.7%, respectively (Table 1). The other 11 taxonomic groups accounted for less than 10% of the total number of endophytes recovered. The diversity and abundance of the endophytes varied according to the type of host tissues. The colonization rate and isolation rate of the endophytic fungi ranged from 32.7% to 100%, and 0.05 to 0.75, respectively. Plant leaves and stems harbored a greater number endophytic fungi than the flowers, with a total of 121 fungal isolates being isolated from the stems, 83 from the leaves, and just 4 from the flowers (Table 2). 3.2. Antifungal activity of endophytic fungi against Blumeria graminis f. sp. tritici Fifteen of the 208 fungal endophytes inhibited Bgt, with rates of inhibition ranging from
65.4% to 100% (Table 3). Three isolates including LPS-1, 16-6 and SCS-6 exhibited much greater activity than the others. Isolate LPS-1 was of particular interest given that it consistently caused 100% inhibition of Bgt colonization (Fig. 1). The results of in vivo seedling assays confirmed the antagonistic activity of the 15 endophytic fungi but revealed a greater degree of variation in their efficacy. In general, the levels of Bgt inhibition were lower than those observed in the detached leaf assay, ranging from just 8.2% to95.0%. Isolate LPS-1 was again the most effective in inhibiting Bgt (85.0%), having more than 20% greater activity than isolates 16-6 (59.6 %) and SCS-6 (63.5%). Indeed, LPS-1 provided significantly better control of Bgt (P< 0.05) than the commercial fungicide triadimefon (10 μg/ml), which only resulted in control efficiencies of 78.2% and 65.8% in the detached leaf and seedling assays, respectively. 3.3. Identification of the endophytic fungus LPS-1 The colony of isolate LPS-1 developed a compact mycelial texture, which was initially white on the 3rd day of incubation (Fig. 2A) but later became dark gray by the 10th day (Fig. 2B). Based on its colony morphology, isolate LPS-1 was tentatively identified as a member of the Botryosphaeriaceae family. Microscopic analysis of the pycnidia, which was formed after 5 weeks of incubation on sterilized pine needles, revealed that the conidia were ellipsoidal in shape, initially hyaline, unicellular (Fig. 2D), becoming dark brown, and developing a thick wall, with central septum, and longitudinal striations with age (Fig. 2E). Mature conidia were 26.3±0.4 to 31.2±0.2 μm long and 11.3± 0.4 to 16±0.7 μm wide (n=60). These morphological characteristics matched previous descriptions of L. pseudotheobromae (Alves et al., 2008). For the molecular identification, the ITS and TEF-1αsequences generated in
this
study
were
deposited
in
GenBank
with
KU180477
and
KU180478,respectively.Polymerase chain reaction (PCR) of the isolates resulted in amplicons of approximately 542 bp for the ITS and 301 bp for the TEF-1αgene regions. Multiple sequence alignment of the LPS-1 sequence with those from other fungi (Fig. 3) revealed that the LPS-1 sequence exhibited 100% sequence identity to a sequence from L. pseudotheobromae, but different from the type strains of L. pseudotheobromae (CBS 116459 and CBS 374.54) by way of nucleotide difference in the TEF-1αgene region. 3.4. Optimal culture conditions for promoting antifungal activity of LPS-1 The most effective medium for promoting antifungal activity of LPS-1 was PDB. No observably difference of Bgt inhibition occurred when LPS-1 was prepared in static PDB and on a rotary shaker (85.1% and 83.8% inhibition, respectively).The static culture was chosen for the following study with the purpose of energy saving.
Many other culture
conditions had significant impact on the antifungal activity of LPS-1 (Fig. 5). Increasing the inoculum load from three mycelial plugs to nine significantly reduced antifungal activity (Fig. 5A), while pH had little impact on Bgt inhibition (Fig. 5B). The optimal temperature for Bgt inhibition was 30°C, with higher or lower temperatures significantly reducing antifungal activity (Fig. 5C). It was also found that the period of incubation significantly affected the inhibition of Bgt, with peak activity occurring at day 6 (Fig. 5D). Taken together these results indicate that the optimal conditions for maximizing the antifungal activity of LPS-1 were to inoculate PDB (pH 7) with three mycelial plugs and to incubate static cultures at 30°C for 6 days. 4. Discussion
Endophytic fungi represent a large portion of fungal species and are ubiquitous in the nature, residing in the intercellular or intracellular spaces of plant tissues for at least a portion of their life cycles without causing apparent symptoms of infection (Petrini et al., 1991). We have obtained and characterized 208 isolates belonging to 17 taxonomic groups of endophytic fungi from 26 Chinese medicinal plants. Most of the isolates (approximately 60%) belong to five taxonomic groups: Phomopsis, Acremonium, Phoma, Alternaria and Colletotrichum. However, many of the isolates recovered from the different plants appeared to have very similar morphology indicating that the same species of endophyte had been repeatedly isolated from different hosts, and that only 40 distinct morphospecies had been collected. As well as exhibiting great taxonomic diversity, some of the endophytic isolates showed a high degree of both host and tissue specificity, which is supported by other reports (Selim et al., 2011; Wiyakrutta et al., 2004; Huang et al., 2008). A large proportion of the isolates collected could not be accurately identified, a common problem with endophytic fungi, and were grouped together within the mycelia sterilia. These isolates were the most abundant accounting for 29.8% of the 208 isolates collected, and found in most of the host plants investigated. The ascomycete fungus L. pseudotheobromae has recently been differentiated from L. theobromae [anamorph: Botryosphaeria rhodina (Berk. & M.A. Curtis) Arx,] based on the differences in its conidial morphology as well as phylogenetic analyses (Alves et al., 2008). Although L. pseudotheobromae is known to be a common pathogen of pantropical trees infecting at least five species in southern China alone, including Acacia confuse, Albizia falcataria, Eucalyptus sp., Mangifera sylvatica and Paulownia fortunei (Zhao et al., 2010), it
has also recently been observed to exhibit an endophytic lifestyle being isolated from the symptomless healthy plant tissues of Camptotheca acuminate (Davidiaceae) (Wei et al., 2014) and llligera rhodantha (Hernandiaceae) (Lu et al., 2 014). To the best of our knowledge, this is the first report of L. pseudotheobromae being isolated from the stem tissues of the medicinal plant Ilex cornuta. The present research was aimed to reduce using chemicals in agriculture process and find out the most suitable non-chemical method to protect wheats against powdery mildew disease. In this study, data showed that LPS-1 culture filtrates significantly reduced disease incidence and severity of wheat powdery mildew. These results might be due to that LPS-1 inhibits disease by producing some antifungal substances. These results are in accordance with many reports that many endophytic fungi produce compounds with antifungal activity (Kusari and Spiteller, 2012; Campos et al., 2008). Endophytic fungi provide an opportunity for the discovery and production of biologically active secondary metabolites. Furthermore, they have considerable advantages over plant sources as manipulation of the fermentation conditions, by altering the medium type and culture conditions (pH, temperature and harvest point), can increase production (Kusari et al., 2012). Investigation of different culture conditions including media, agitation, inoculum load, pH and temperature as well as the duration of incubation revealed considerable variation in the antagonistic activity of LPS-1 against Bgt. The optimal conditions resulted from inoculating PDB (pH 7.0) and incubation at 30°C for 6 days without shaking. Under these conditions LPS-1 exhibited better antifungal activity in both the detached leaf and seedling assays than a 10 μg/ml application of the commercial fungicide triadimefon (Table 3), which suggests that LPS-1 has the potential to
be an excellent antimicrobial agent for the control of wheat powdery mildew. Having established the optimal fermentation conditions, further research is required to identify the bioactive compound(s) responsible for the antifungal activity of LPS-1. Endophytic fungi and their host plants coevolved over a long period of time; therefore, many plants that produce bioactive products have associations with endophytic fungi capable of producing the same compounds, perhaps as a consequence of their adaption to such a specific environment (Luiz et al., 2011). For example, the famed anticancer diterpenoid paclitaxel, which occurs in all yew species (Taxus sp.) (Suffness and Wall, 1995), is also produced by the fungus Taxomyces andreanae Strobel, Stierle & Hess, which has been isolated from the pacific yew Taxus brevifolia Nutt. (Strobel et al., 1993). The medicinal plant I. cornuta also appears to be a promising source of endophytic microorganisms capable of producing metabolites with proven antimicrobial activity (Wu et al., 2014). It is therefore interesting to note that recent studies have shown that L. pseudotheobromae can produce many bioactive compounds with medicinal properties (Wei, et al., 2014; Lu, et al., 2014). For example, it was found that L. pseudotheobromae isolate F2, which was isolated from Camptotheca
acuminate
(Davidiaceae),
produced
six
new
sulfur-containing
diketopiperazines named Lasiodipline 1-6, and that Lasiodipline 5 was confirmed to be a novel antiprotozoal compound with potency comparable to the commercial drug tinidazole (Wei et al., 2014). Similar investigation of L. pseudotheobromae isolate XSZ-3, which was isolated from the branches of Camptotheca acuminate, was found to produce four different palmarumycins, one of which was characterized as a novel anticancer compound palmarumycin LP1, which exhibited significant inhibitory activity against human
promyelocytic leukemia cells with an IC50 value of 2.39 μM. (Lu et al., 2014). These results confirm that L. pseudotheobromae has great potential as a source of novel compounds for the pharmaceutical industry. Considering that other isolates such as F2 and XSZ-3 have yielded a range of bioactive compounds it is likely that isolate LPS-1, which was isolated from the medicinal plant I. cornuta, could also be a valuable resource, and the results of the current study suggest that these may not be restricted to clinically important compounds but also agriculturally important ones.In conclusion, various endophytic fungi were harbored in traditional Chinese medicinal plants, and some of them were strong antagonists. Their representative isolates exhibited significant antifungal activities against Bgt. To the best of our knowledge this represents the first report of L. pseudotheobromae having antifungal activity against plant pathogens. Isolate LPS-1 can be potential biocontrol agent, and therefore has been selected for further research in an ongoing project.
Acknowledgments This research was supported by the MATS grant from the Department of Agriculture (CARS-03-04B), funded by the R&D Special Fund for Public Welfare Industry (Agriculture) of China (Grant Nos.201303016, 201303025), the Hubei Province Science and Technology Innovation Center (2011-620-003-3) and the Foundation for Youths of Hubei Academy of Agricultural Sciences (2015nkyjj13).
References Alves, A., Crous, P.W., Correia, A., Phillips, A.J.L., 2008. Morphological and molecular data reveal cryptic speciation in Lasiodiplodia theobromae. Fungal Divers. 31, 1-13. Aly, A.H., Debbab, A., Kjer, J., Proksch, P., 2010. Fungal endophytes from higher plants: a prolific source of phytochemicals and other bioactive natural products. Fungal Divers. 41, 1–16. Backman, P.A., Sikora, R.A., 2008. Endophytes: an emerging tool for biological control. Biol. Control 46, 1–3. Campos, F., Rosa, L., Cota, B., Caligiorne, R., Rabello, A., Alves, T., Rosa, C., Zani, C., 2008. Leishmanicidal metabolites from Cochliobolus sp., an endophytic fungus isolated from Piptadenia adiantoides (Fabaceae). PLoS Negl Trop Dis. 2, e348. Carbone, I., Kohn, L.M., 1999. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia. 91:553–556. Crous, P.W., Slippers, B., Wingfield, M.J., Rheeder, J., Marasas, W.F.O., Philips, A.J.L., Alves, A., Burgess, T., Barber, P., and Groenewald, J.Z., 2006. Phylogenetic lineages in the Botryosphaeriaceae. Stud Mycol. 55, 235–253. Curtis,F.d.,Cicco.V.D.,lima,G.,2012.Efficacy of biocontrol yeasts combined with calcium silicate or sulphur for controlling durum wheat powdery mildew and increasing grain yield components. Field Crops Res. 134, 36-46. Elad,Y., Malathrakis,N. E., Dik,A. J., 1996. Biological control of botrytis-incited diseases and powdery mildews in greenhouse crops. Crop Protect. 15, 229–240. Gao, X.N, Gong Y.F., Huo, Y.X., Han,Q.M., Kang, Z.S., Huang,L.L.,2015 Endophytic bacillus subtilis strain E1R-J is a promising biocontrol agent for wheat powdery mildew. BioMed Res.Int.2015,
462645-462652. Gong S.J., Yang L.J.,Xiang L.B., Zeng F.S., Zhang X.J., Xue M.F.,Wang H.,Shi W.Q., Yu D.Z. 2013 Baseline sensitivity of Blumeria graminis f．sp．tritic to metrafenone，quinoxyfen and bioactivitives of these fungicides. Chin. J Pestici .Sci. 15,511-515. Hijri, M., Redecker, D., Jean, A., Petetot, M.C., Voigt, K., Wöstemeye, R.J., Sanders, I.R., 2002. Identification and isolation of two ascomycete fungi from spores of the arbuscular mycorrhizal fungus Scutellospora castanea. Appl. Environ. Microbiol. 68, 4567–4573. Hallmann, J., Berg, G., Schulz, B., 2006. Isolation procedures for endophytic microorganisms. In: Schulz, B.J.E., Boyle, C.J.C., Sieber, T.N. (Eds.), Microbial Root Endophytes. Springer, Berlin-Heidelberg, pp. 299–319. Huang, W.Y., Cai, Y.Z., Hyde, K.D.,Corke, H., Sun, M., 2008. Biodiversity of endophytic fungi associated with 29 traditional Chinese medicinal plants. Fungal Divers. 33, 61-75. Ismail, A.M., Cirvilleri, G., Polizzi, G., Crous, P.W., Groenewald, J.Z., Lombard, L., 2012. Lasiodiplodia species associated with dieback disease of mango (Mangifera indica) in Egypt. Austral. Plant Pathol. 41, 649-660. Ji, H.F., Li, X.J., Zhang, H.Y., 2009. Natural products and drug discovery. Can thousands of years of ancient medical knowledge lead us to new and powerful drug combinations in the fight against cancer and dementia? EMBO Rep. 10, 194–200. Kharwar, R.N., Mishra, A., Gond, S.K., Stierle, A., Stierle, D. 2011. Anticancer compounds derived from fungal endophytes: their importance and future challenges. Nat. Prod. Rep. 28, 1208–1228.
Kiss, L.,2003. A review of fungal antagonists of powdery mildews and their potential as biocontrol agents. Pest Manage Sci.59, 475–483.
Kusari, S., Spiteller, M., 2012. Metabolomics of endophytic fungi producing associated plant secondary metabolites: progress, challenges and opportunities. In Metabolomics, U. Roessner, ed. (Rijeka, Croatia: In Tech), pp. 241–266. Kumar, S., Kaushik, N.,2013, Endophytic fungi isolated from oil-seed crop jatropha curcas produces oil and exhibit antifungal activity. PLoS One 8,e56202. Kusari, S., Hertweck, C., Spiteller, M., 2012, Chemical ecology of endophytic fungi: origins of secondary metabolites. Chem Biol. 27, 792-798. Liu W C, Shao Z R. 1994. The epidemic tendency of wheat powdery mildew in China. In: Li G B, ed., Prospects of
Plant
China, Beijing.
Protection
Research
in
China.
Science
and
Technology
Press
of
pp.135-141. (in Chinese)
Lu, X.J., ChenG., Li, Z.L., Zhang, Y.H., Wang, Z.Y., Rong, W.W., Pei, Y.H., Pan, H.Q., Hua, H.M., Bai J., 2014. Palmarumycins from the Endophytic Fungus Lasiodiplodia pseudotheobromae XSZ-3. Helvetica. Chimica. Acta. 97, 1289-1294. Luiz, H. R., Mariana, L.A., Vieira, B.B., Cota, S.J., T nia, M.A., Alves, Clos L.Z., Carlos A.R., 2011. Endophytic Fungi of Tropical Forests: A Promising Source of Bioactive Prototype Molecules for the Treatment of Neglected Diseases, Drug Development -A Case Study Based Insight into Modern Strategies, Rundfeldt, C., (Ed.), In Tech, Rijeka. pp.469-486. Petrini, O., Fungal endophytes of tree leaves. 1991. In: Andrews, N.J., Hirano, S.S., (eds), Springer, New York. pp. 179–197. Selim K.A., El-Beih, A.A., AbdEl-Rahman, T.M., El-Diwany, A.I., 2011. Biodiversity and antimicrobial activity of endophytes associated with Egyptian medicinal plants. Mycosphere 6, 669-678. Staniek, A., Woerdenbag, H.J., Kayser, O., 2008. Endophytes: exploiting biodiversity for the improvement
of natural product-based drug discovery. J. Plant Interact. 3, 75–93. Strobel, G. A., Li, J.Y., Sugawara, F., Koshino, H., Harper, J., Hess., W.M., 1999. Oocydin A, a chlorinated macrocyclic lactone with potent anti-oomycete activity from Serratia marcescens. Microbiol. 145, 3557–3564. Strobel, G. A., Stierle, A., Stierle, D., Hess, W.M., 1993. Taxomyces andreanae, a proposed new taxon for a bulbilliferous hyphomycete associated with Pacific yew (Taxus brevifolia). Mycotaxon. 47, 71–78. Strobel, G.A., Daisy, B., Castillo, U., Harper, J., 2004. Natural products from endophytic microorganisms. J. Nat. Prod. 67, 257–268. Suffness, M., Wall, M.E., 1995. Discovery and development of taxol, In Taxol: science and applications. Suffness, M. (Ed.), Taxol: Science and Applications. CRC Press, Boca Raton, Florida. pp. 1–25 Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596-1599. Vieira, M.L., Johann, S., Hughes, F.M., Rosa, C.A., Rosa, L.H., 2014. The diversity and antimicrobial activity of endophytic fungi associated with medicinal plant Baccharis trimera (Asteraceae) from the Brazilian savannah. Can. J. Microbiol. 60, 847-856. Vilgalys, R., Hester, M., 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J. Bacteriol. 172, 3238-4276. White, T.J., Bruns, T, Lee, S., Taylor, J.W., 1990. Sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J., (eds.) PCR Protocols: a guide to methods and applications. Academic Press, Inc., New York. pp. 315-322 Winter,J., M. , Behnken,S., Hertweck,C., 2011. Genomics-inspired discovery of natural products. Current Opinion in Chemical Biology, 15,22–31
Wiyakrutta, S., Sriubolmas, N., Panphut, W., Thongon, N., Danwiserkanjana, K., Ruangrungsi, N., Meevootisom, V., 2004. Endophytic fungi with antimicrobial, anti-cancer, anti-malarial activities isolated from Thai medicinal plants. World. J. Microb. Biot. 20, 265-272. Wei, W., Jiang, N., Mei, Y.N., Chu, Y.L., Ge, H.M., Song, Y.C., Ng, S.W., Tan, R.X., 2014. An antibacterial metabolite from Lasiodiplodia pseudotheobromae F2. Phytochemistry 100,103-109 Wu, Y., Zhang, X.C., Sun, J.B., Wu, X., Li, Y.Y., Gu, W., Wang, D.Y., Liu, J.G., Hu, Y.L., 2014. Chemical constituents from the leaves of Ilex urceolatus, Biochem. Syst. Ecol. 54, 92-95. Yang L., Zhou Y. L., Duan X. Y., Cao X. R., Zou Y. F., 2011.Sensitivity of Blumeria graminis f. sp. tritici isolates to triadimenfor and fenpropidin in China. Acta Phytopathol Sinica, 43, 430–434.Yang, X.J., Yang, L.J., Wang, S.N., Yu, D.Z., Ni, H.W., 2008. Effect of physcion, a natural anthraquinone derivative, on the infection process of Blumeria graminis on wheat. Can. J. Plant Pathol. 30, 391-396. Zhang, J., Wu, M.D., Li, G.Q., Yang, L., Yu, L., Jiang, D.H., Huang, H.C., Zhuang, W.Y., 2010. Botrytis fabiopsis, a new species causing chocolate spot of broad bean in central China. Mycologia. 102, 1114–1126. Zhang, Q.H., Zhang, J., Yang, L., Zhang, L., Jiang, D.H., Chen., W.D., Li, G.Q.,2014.Diversity and biocontrol potential of endophytic fungi in Brassica napus. Biol. Control 72, 98-108. Zhao, J.P., Lu, Q., Liang, J., Decock, C., Zhang, X.Y., 2010. Lasiodiplodia pseudotheobromae, a new record of pathogenic fungus from some subtropical and tropical trees in southern China. Cryptogamie Mycol. 31, 431-439.
Fig. 1. Effect of LPS-1 on the proliferation of Blumeria graminis f.sp. tritici. A, Detached leaf assay; B, Wheat seedling assay.
Fig. 2. Colony and conidial morphology of LPS-1. A, Colony morphology after 3 days incubation at 25°C; B, Colony morphology after 10 days at 25°C; C, Conidiogenous cells and young conidia; D, Hyaline, aseptate conidia. E, Septate, dark-walled conidia. The conidia micrographs were taken at ×100 magnification (oil immersion) from pycnidia formed on pine needles. Scale bar = 10 µm.
Fig. 3. Phylogenetic tree based on maximum-likelihood analysis of combined ITS and TEF-1α sequence data. Bootstrap
values >51% are presented above branches, and bootstrap values <51% are not shown. Guignardia philoprina (CBS447.68) represents the out-group.
Fig. 4. Effect of different media on the antifungal activity of LPS-1. Liquid cultures (pH 7.0) were inoculated with three mycelial plugs (5 mm in diameter) and incubated at 30℃ for 6 days: PDB, potato dextrose broth; MPDB, modified potato dextrose broth; MEB, malt extract broth; and CDB, Czapek-Dox broth.
Fig. 5. Effect of inoculum load, pH, temperature and duration of incubation on the antifungal activity of LPS-1.
Table 1 Number of endophytic fungi belonging to different taxa isolated from 26 Chinese medicinal plants Plant host\Fungal taxa
a
C
A
Ph
P
F
A
M
C
B
G
X
P
C
P
Ph
R
S
Tota
o
l
o
h
u
c
s
h
o
u
y
l
l
e
y
h
c
l
Eomecon chionantha Xylosma racemosum
1 3
1 4
Fraxinus hupehensis
5 1
1
4
Silybum marianum
marginatum Euonymus alatus
Epimedium sagitlatum
3
2
2
Buddleja lindleyana
3
4
5
3
3
3
1
1
1
10
1
4
12
1
8 2
1
4
7 1
4
2
1
Podocarpus
1
3
Nandira domestica
2
3
2
3 2
1
Narcissus
2
pseudonarcissus Syringa tomentella
16 2
6
3
3
3
5
5
16
5
9
2
5
3
8
2
3
9
1
1
16
2
Polygonum
1 1
capitatum Pinus massoniana
2 16
1 9
1 39
3 2 2
6
3
4
2
Abelia macrotera
1
4 3
2
macrophyllus
Total
13 1
1
bachli
macrolepis
8
4
2
Rhododendron
Calocedrus
1
2
Torreya grandis
chindensensevar
3
2
Papaver rhoeas
Lorpetalum
10
7
2 5
1
1
4
4
Buxus ichangensis
Peristrophe japonic
1
4
2
3
Bupleurum
Ilex cornuta
2
1
1
Buxus bodiniero
14 3
3 1
1
2
Callicarpa bodinieri Saxifraga stdonifera
1
3
2
3
3
3
12
24
62
4
4
1
3
2
1
1
1
1
2
208
a Morphologically identified fungal taxa: Fu: Fusarium spp.; Al: Alternaria spp.; Ph: Phoma spp.; Pho: Phomopsis spp.; Co: Colletotrichum spp.; Pl: Pleosporales spp.; MS: mycelia sterilia spp.; Ch: Chaetomium spp.; Bo: Botryosphaeria spp.; Gu: Guignardia spp.; Xy: Xylariales spp.; Ac: Acremonium spp.; Cl: Cladosporium spp.; Pe: Penicillium spp.; Phy: Physalospora spp.; Rh: Rhizosphaera spp.; SC: Scopulariopsis spp.;
Table 2 Number of endophytic fungi isolated from the stems, leaves and flowers of 26 Chinese medicinal plants growing in the Wuhan botanical gardens at the Chinese Academy of Sciences.
Number of fungal isolates
Plant source
Stem
Leaf
Flower
Total
Eomecon chionantha
NIa
1
NI
1
Xylosma racemosum
6
8
NI
14
Fraxinus hupehensis
NI
3
NI
3
Callicarpa bodinieri
4
NI
NI
4
Saxifraga stdonifera
7
2
NI
9
Silybum marianum
3
NI
NI
3
Buxus bodiniero
NI
7
NI
7
Bupleurum marginatum
8
NI
NI
8
Euonymus alatus
NI
13
NI
13
Ilex cornuta
6
NI
4
10
Epimedium sagitlatum
5
6
NI
11
Buddleja lindleyana
5
6
NI
11
Buxus ichangensis
NI
2
NI
2
Papaver rhoeas
NI
7
NI
7
Torreya grandis
NI
1
NI
1
Peristrophe japonic
NI
17
NI
17
Rhododendron bachli
1
5
NI
6
Podocarpus macrophyllus
8
1
NI
9
Lorpetalum chindensensevar
6
9
NI
15
Calocedrus macrolepis
7
11
NI
18
Nandira domestica
2
7
NI
9
Abelia macrotera
2
3
NI
5
Narcissus pseudonarcissus
6
2
NI
8
Syringa tomentella
NI
2
NI
2
Polygonum capitatum
1
2
NI
3
Pinus massoniana
6
6
NI
12
Total
83
121
4
208
NI = not isolated.
Table 3 Antifungal activity of selected isolates of endophytic fungi against Blumeria graminis f.sp. tritici on detached leaves and seedlings of wheat.
Detached leaves assay Treatment
Whole seedling assay
Severity1
Efficacy2
Severity
Efficacy
ESL-3
18.1±5.9efg
80.2±5.8
34.6±5.0h
57.1±5.9
LPS-1
0.0i
100
14.0±3.6i
85.0±4.9
16-6
8.7±3.2h
90.4±3.7
32.5±3.5h
59.6±5.8
PBP-4
30±4.4bc
67.0±4.2
45.7±3.4g
43.2±6.1
LPS-4
24.4±4.1bcde
73.2±3.5
52.8±7.1efg
33.0±8.8
16-3
14.4±6.0fgh
84.2±6.3
55.7±6.3def
31.1±6.4
SCS-4
26.4±4.8bcd
70.9±5.3
62.7±3.1cde
22.1±6.6
SCS-5
25.6±7.1bcd
71.6±8.9
69.0±3.0bc
14.5±1.7
SCS-6
7.9±1.0h
91.3±0.8
30.8±2.3h
63.5±2.7
EML-3
11.4±2.1gh
87.4±2.5
56.3±9.4def
30.3±9.6
BDS-3
22.5±1.7cde
75.2±2.8
70.7±5.1bc
12.4±4.8
LPF-4
29.7±6.3bc
67.4±6.1
58.8±5.9def
27.1±6.9
LKL-8
31.4±2.1b
65.4±1.9
74.1±5.0ab
8.2±2.9
PLS-1
29.7±3.4bc
67.1±4.9
64.7±6.9cd
19.9±6.9
PLS-3
17.2±1.7efg
81.0±2.0
51.3±5.5fg
36.2±8.6
CK2
19.7±2.1def
78.2±3.2
27.5±2.8h
65.8±4.7
CK1
90.8±3.8a
CK1, Negative control treated with PDB and inoculated with Bgt.
80.7±3.3a
CK2, Positive control treated with 10 μg/ml triadimefon and inoculated with Bgt. 1
Means followed by the same letters within a column do not differ significantly (P>0.05) according to Duncan’s Multiple
Range Test. 2
The biocontrol efficacy was calculated using the following formula: biocontrol efficacy (%) = (negative control
treated−treated)/ negative control treated×100%. The disease severity was investigated 12 days after the inoculation of Blumeria graminis f. sp. tritici.
Highlights
208 endophytic fungal isolates were collected from stems (83), leaves (121) and flowers (4) of 26 medicinal plants.
Fifteen endophytic fungi exhibited antifungal activity.
Strain of L. pseudotheobromae has strong biological control of wheat powdery mildew.
The first report of L. pseudotheobromae having antifungal activity against plant pathogens.
Graphical abstract
S1049-9644(15)30058-X http://dx.doi.org/10.1016/j.biocontrol.2015.12.002 YBCON 3356
To appear in:
Biological Control
Received Date: Revised Date: Accepted Date:
19 August 2015 25 November 2015 4 December 2015
Please cite this article as: Xiang, L., Gong, S., Yang, L., Hao, J., Xue, M., Zeng, F., Zhang, X., Shi, W., Wang, H., Yu, D., Biocontrol potential of endophytic fungi in medicinal plants from Wuhan Botanical Garden in China, Biological Control (2015), doi: http://dx.doi.org/10.1016/j.biocontrol.2015.12.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Biocontrol potential of endophytic fungi in medicinal plants from Wuhan Botanical Garden in China Libo Xianga, Shuangjun Gonga, Lijun Yanga, Jianjun Haob, MinFeng Xuea, FanSong Zenga, XueJiang Zhanga, WenQi Shia, Hua Wanga ,Dazhao Yua a
Institute of Plant Protection and Soil Science, Hubei Academy of Agricultural Sciences, Key
Laboratory of Integrated Pest Management on Crop in Central China, Ministry of Agriculture, Hubei Key Laboratory of Crop Diseases, Insect Pests and Weeds Control, Wuhan 430064, China; b
School of Food and Agriculture, The University of Maine, Orono, ME 04469, USA
These authors contributed equally to this work. Corresponding author. E-mail address: [email protected],
Abstract Many medicinal plants possess antimicrobial activities, and have antagonistic endophytic fungi that help them protect from pathogen attack. The aim of this study was to examine endophytic fungi in traditional Chinese medicinal plants, and understand if these organisms have antimicrobial activities and they can be potentially used for biological control of plant diseases. A total of 208 endophytic fungal isolates were collected from stems (83), leaves (121) and flowers (4) of 26 medicinal plant species. The majority of the isolates belonged to Alternaria, Phomopsis, Colletotrichum, Phoma and Acremonium as well as several species allocated to mycelia sterilia. A detached leaf assay was conducted by testing these isolates on wheat powdery mildew (Blumeria graminis f.sp. tritici, or Bgt). Fifteen isolates of endophytic fungi inhibited Bgt, exhibiting control efficacies ranging from 65.4 to 100%. Of these isolates, LPS-1, SCS-6 and 16-6, exhibited significant inhibition of Bgt proliferation (>90%). Isolate LPS-1 isolated from the stem of Ilex cornuta Lindl. ex Paxt. had the highest efficacy, resulting in 100% inhibition of Bgt growth on detached leaf segments. Based on morphological characteristics and phylogenetic analyses of the ITS rDNA sequences and translation elongation factor 1-alpha (TEF-1α)
gene regions,
LPS-1 was
identified as Lasiodiplodia pseudotheobromae. A range of culture conditions for LPS-1 were examined and the results indicated that optimal antifungal activity resulted from static cultures in PDB (pH 7.0) inoculated with three mycelial plugs and incubated at 30°C for 6 days. With further study, LPS-1 can be a candidate for biological control. Keywords: Medicinal plants; Endophytic fungi; Lasiodiplodia pseudotheobromae; Biological control
1 Introduction Endophytic fungi play an important role in enhancing plant health, and have been recognized as an important resource of biocontrol agents to suppress plant pests including both insects and pathogens (Backman and Sikora, 2008; Kumar and Kaushik ,2013;Zhang et al.,2014). Medicinal plants could be a valuable repository for fungal endophytes yielding novel metabolites of agricultural and pharmaceutical importance (Kusari and Spiteller, 2012; Winter et al., 2011; Ji et al., 2009). In Brazil, for example, medicinal plant Baccharis trimera (Asteraceae)
harbores endophytic fungi, belonging to 25 different taxa (Vieira et al., 2014),
and exhibited a broad spectrum of inhibitory activity against a range of pathogenic bacteria. Furthermore, endophytes synthesizing bioactive metabolites can be used directly or indirectly as biocontrol agents against plant diseases (Strobel et al., 2004; Staniek et al., 2008; Aly et al., 2010; Kharwar et al., 2011; Kusari and Spiteller, 2012). One endophytic isolate of Cochliobolus sp. obtained from the plant Piptadenia adiantoides (Fabaceae) produces antifungal compounds cochlioquinone A and isocochlioquinone A (Campos et al., 2008). As a consequence the study of endophytic fungi has received more and more attention. Powdery mildew (Blumeria graminis Speer) is a severe disease, causing significant economic losses in a wide range of field crops. In China, the disease affected up to 12 million hectares in 1990 and resulted in estimated grain yield losses of 14.4 million metric tons (Liu and Shao 1994). Over the last decade, an average of more than six million hectares of growing area has been affected annually (http://www.agri.gov.cn/). To date management of this disease has largely been reliant on the application of chemical fungicides such as sterol demethylation inhibitors (DMIs). However, the development of pathogen strains resistant to
the mostly used triadimefon fungicide has been reported and the resistant strain is now widespread (Yang et al., 2011; Gong et al., 2013).
Alternatively, finding strategies such as
biocontrol for the management of this devastating disease are urgently required. Many reports have been found to be effective as biocontrol agents against various powdery mildew fungi (Elad et al., 1996; Kiss, 2003; Gao et al., 2015), but little research has been conducted on control of wheat powdery mildew using BCAs. The objectives of this study were to explore the diversity of endophytic fungi associated with traditional Chinese medicinal plants, to screen the isolates obtained for antagonistic activity with the potential to control Bgt, and third to optimize the culture conditions of candidate biocontrol agents to maximize their antifungal activity. 2. Materials and methods 2.1. Isolation of endophytic fungi Tissues of stems, leaves and flowers were collected from healthy medicinal plants at the Wuhan Botanical Garden, Chinese Academy of Sciences. The leaflets, stem and flower segments were surface sterilized according to the protocol of a previous study (Hallmann et al., 2006). The plant material was thoroughly washed using distilled water, followed by treatment with 70% ethanol for 2 min and 5% sodium hypochlorite for 5 min and then rinsed in sterile distilled water. The water from the final wash was collected and plated onto PDA as a positive control to confirm that the surface sterilization had been successful. The sterilized samples were then cut into 5 to 6 pieces (2 to 6 mm in diameter) and plated onto water agar (distilled water, 1.5% agar) amended with 100 μl/ml ampicillin. The samples were then incubated at 25°C for 3 to 14 days until fungal growth was observed on their surfaces (Hijri
et al., 2002). The emerging hyphal tips were then picked and purified by successive subculture on potato dextrose agar (PDA) plates before being maintained as stock cultures at 4°C. 2.2. Identification and diversity of endophytic fungi The isolates of endophytic fungi were provisionally identified under a microscope based on the morphology of their colonies (color and mycelial characteristics) and spores (conidia, blastospores, sporangiospores or ascospores). They were confirmed by analyzing the sequence of the internal transcribed spacer (ITS) region of their rDNA (ITS1-5.8S rDNA-ITS2),
which
were
amplified
using
the
universal
primers
ITS1
(5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) (White et al., 1990). The resulting PCR products were then cloned and sequenced according to the protocol of a previous study (Zhang et al., 2010). Cultures that failed to sporulate were grouped as mycelia sterilia, and divided into different morphospecies according to their culture characteristics. The colonization rate (CR) for each plant species was calculated as the total number of plant tissue pieces colonized by endophytes divided by the total number of pieces incubated for each plant sample expressed as a percentage, while the isolation rate (IR), which is a measure of endophyte richness of a given sample of plant tissue, i.e. the number of multiple colonization by different endophytes in a single tissue sample, was calculated as the number of isolates obtained divided by the total number of tissue pieces, but not expressed as a percentage (Huang et al., 2008). 2.3. Assay for antagonistic activities of endophytic fungi on detached leaf and wheat seedling
Culture filtrates from the endophyte isolates were prepared as follows: mycelial plugs from each isolate were inoculated in 100 ml potato dextrose broth (PDB: 200g/l potato and 20 g/l sucrose) in a 250 ml Erlenmeyer flask and incubated with shaking (150 rpm) at 28°C for 7 days. The resulting cultures were then centrifuged at×8000g at 4°C, filtered through 0.22 μm membrane filters (EMD Millipore Corporation, Billerica, MA, USA) to remove hyphal fragments and stored at 4°C until required. A total of 208 isolates were assessed for their antifungal activity against Bgt using a detached leave assay described in a previous study (Yang et al. 2008). Leaf segments (2.5 cm in length) were cut from wheat plants (cv. Chancellor) at the two-leaf stage and placed randomly on 9 cm petri dishes containing 0.5% benzimidazole water agar (w/v). The culture filtrates (4 mL) were then applied to the plates using a potter spray tower (model PDE0012, Burkard Scientific Ltd, Middlesex, UK). Bgt conidia were produced by inoculating detached, surface-sterilized leaves of wheat cv. Chancellor, and then placed in a growth chamber at 18±1◦C and constant light. After air-drying for 12 h, the treated plates were inoculated at a density of 2-4×103 conidia/cm2 in a setting tower and incubated in a growth chamber with a 12 h photoperiod and 70% relative humidity. Negative and positive controls were prepared for each endophyte isolate using sterile PDB media and 10 μg/ml triadimefon, respectively, which were similarly applied using the potter spray. The disease severity was evaluated at 12 days post inoculation (dpi) using the method described in a previous study (Curtis et al., 2012) and the following scale:
0=no visible pathogen
colonization of the leaves; 1=1–5%, 2=6–10%, 3=11–25%, 4=26–50%, 5=51–75% and 6=76–100% of leaf area covered by the pathogen. Each treatment was represented by three
replicate dishes each containing ten leaf segments, and the entire experiment was conducted twice. After preliminary screening using the leaf segment assay, 15 endophytic isolates exhibiting antifungal activity were selected for further investigation using a wheat seedling assay. Fifteen wheat seeds (cv. Chancellor) were sown in pots (10 cm in diameter) containing soil amended with 0.5% (w/w) of a commercial fertilizer (N: P: K = 15:15:1; Hubei Dong Sheng Chemicals Group Co., Ltd., Yuan An County, Hubei, China). The pots were placed in plastic boxes with a shallow water level to maintain high humidity and kept in a growth chamber at 18°C with a 12 h light/12 h dark photoperiod until the emerged seedling were ten days old. Culture filtrates from the endophytic fungi were then sprayed on the seedlings, which were allowed to air dry for approximately 24 h before being inoculated with the equivalent of 2-4×103 conidia/cm2 using a miniature settling tower, consisting of a 2 L cup with a 1.5 cm opening in its top to allow aerial distribution of the test conidia onto the leafs located on the plants below. The seedlings were then placed in a growth chamber and maintained under the same conditions as described above, before the disease severity was evaluated at 12 dpi using the 0-7 scale from the detached leaf assay. The experiment had a completely randomized design, using three replicates for each endophyte isolate and negative and positive controls. The commercial fungicide triadimefon as positive control (96 % active, Jiangsu Runfeng Agrochemicals Co., Ltd., Jiangsu, China) prepared in accordance with the detached leaf assay described above. The experiments were performed twice and three replicates were used in each experiment. Data from repeated experiments were pooled before statistical analyses.
2.4. Identification of the endophytic fungus LPS-1 A preliminary identification of isolate LPS-1 was made by examining its colony characteristics, and microscopic analysis of its hyphal and conidial morphology. Sporulation was induced by culture on 2% water agar (WA) on top of which sterilized pine needles had been placed and incubation at 25°C for 5 weeks under near UV light (Ismail et al., 2012). The morphology (cell wall, shape, color, and presence or absence of septa) of the conidia within the pycnidia was assessed using a compound microscope (Nikon Inc. Instruments Group, Elville, NY). Having established that LPS-1 was likely to belong to the genus Lasiodiplodia, specifically
Lasiodiplodia
pseudotheobromae.Multigene
phylogenetic
analyses
were
conducted based on DNA sequence comparisons of the ITS rDNA sequences and translation elongation factor 1-alpha (TEF-1a) gene region (Vilgalys and Hester, 1990). Before conducting the phylogenetic analyses, DNA extraction, DNA amplification and DNA sequencing of the selected isolates were performed using the method described in zhang et al(2010). he ITS and TEF-1αsequences. The sequences of the ITS and TEF-1αgene regions were deposited in GenBank (http://www.ncbi.nlm.nih.gov) The sequences of the type specimen strains that were related to the fungus isolated in this study were obtained from GenBank. Sequence alignment and manual editing of the sequence data were performed using the Bioedit software package (http://www.mbio.ncsu.Edu/BioEdit/bioedit.html). Each of the ITS, TEF-1αand the combined ITS and TEF-1αdatasets was analyzed using the maximum-likelihood (ML) method using Molecular Evolutionary Genetics Analysis (MEGA) version 4.0 software (Tamura et al., 2007). Guignardia philoprina
(CBS446.783) was used as the out-group taxon. 2.5. Optimization of medium and culture conditions to maximize the antifungal activity of LPS-1 The optimal medium for the antifungal activity of LPS-1 as well as the effect of static or rotary culture was determined using four different media: PDB, modified potato dextrose broth (MPDB: 200 g/l potato, 20 g/l sucrose, 19.95 g/l malt extract, 0.5 g/l MgSO•7H2O, and 7.5 g/l NaNO3), malt extract broth (MEB: 20 g/l sucrose, 19.95 g/l malt extract, 0.5 g/l MgSO •7H2O, and 7.5 g/l NaNO3), and and Czapek-Dox broth (CDB: 30 g/l sucrose, 1 g/l KH2PO4, 0.5 g/l MgSO•7H2O, 3 g/l NaNO3, 0.5 g/l KCl, and 0.01 g/l FeSO4•7H2O). Liquid cultures (pH 7.0) were prepared by inoculating 100 ml medium with three mycelial plugs (5 mm in diameter) and incubating at 30°C for six days. The culture filtrates were then collected and assessed using the detached leaf assay described above (2.4). The optimal timing of antifungal activity was also evaluated using PDB liquid cultures as described above and seven different time points: 3, 4, 5, 6, 7, 8, and 9 days. Similar experiments were then conducted to assess the effect of inoculum load (3, 4, 5, 6 and 7 mycelial plugs) and pH (6.0, 6.5, 7, 7.5, and 8), both of which involved 6 days incubation at 28°C, as well as the effect of temperature (20, 25, 30, 35, and 40°C). The experiments were performed twice and three replicates were used in each experiment. Data from repeated experiments were pooled before statistical analyses. 2.6. Data analysis Data were analyzed using the SAS software package (SAS Institute, Cary, NC, USA, Version 8.0, 1999). Analysis of variance was performed, and means of treatment effects were
seperated using Duncan’s multiple range test at the significance level α=0.05. In order to compare the treatments with those applied separately, disease severity was transformed into percentages of control efficacy as follows: Efficacy = (C−T)/C] × 100, where C is the disease severity in the control (treated with PDB medium) and T is the disease severity in the examined treatment 3. Results 3.1. Diversity of endophytic fungi from Chinese medicinal plants A total of 208 endophytic fungal strains were isolated from the 26 traditional Chinese medicinal plants. A total of 40 different morphospecies were identified belonging to 17 taxonomic groups. The majority of the isolates were classified into six groups (Table 1) with the highest number being allocated to the mycelia sterilia (29.8%). The second most abundant group was the Phomopsis, which represented 18.8% of the isolates, followed by the Acremonium, Phoma, Alternaria and Colletotrichum, which had relative frequencies of 11.5%, 10.6%, 9.1% and 7.7%, respectively (Table 1). The other 11 taxonomic groups accounted for less than 10% of the total number of endophytes recovered. The diversity and abundance of the endophytes varied according to the type of host tissues. The colonization rate and isolation rate of the endophytic fungi ranged from 32.7% to 100%, and 0.05 to 0.75, respectively. Plant leaves and stems harbored a greater number endophytic fungi than the flowers, with a total of 121 fungal isolates being isolated from the stems, 83 from the leaves, and just 4 from the flowers (Table 2). 3.2. Antifungal activity of endophytic fungi against Blumeria graminis f. sp. tritici Fifteen of the 208 fungal endophytes inhibited Bgt, with rates of inhibition ranging from
65.4% to 100% (Table 3). Three isolates including LPS-1, 16-6 and SCS-6 exhibited much greater activity than the others. Isolate LPS-1 was of particular interest given that it consistently caused 100% inhibition of Bgt colonization (Fig. 1). The results of in vivo seedling assays confirmed the antagonistic activity of the 15 endophytic fungi but revealed a greater degree of variation in their efficacy. In general, the levels of Bgt inhibition were lower than those observed in the detached leaf assay, ranging from just 8.2% to95.0%. Isolate LPS-1 was again the most effective in inhibiting Bgt (85.0%), having more than 20% greater activity than isolates 16-6 (59.6 %) and SCS-6 (63.5%). Indeed, LPS-1 provided significantly better control of Bgt (P< 0.05) than the commercial fungicide triadimefon (10 μg/ml), which only resulted in control efficiencies of 78.2% and 65.8% in the detached leaf and seedling assays, respectively. 3.3. Identification of the endophytic fungus LPS-1 The colony of isolate LPS-1 developed a compact mycelial texture, which was initially white on the 3rd day of incubation (Fig. 2A) but later became dark gray by the 10th day (Fig. 2B). Based on its colony morphology, isolate LPS-1 was tentatively identified as a member of the Botryosphaeriaceae family. Microscopic analysis of the pycnidia, which was formed after 5 weeks of incubation on sterilized pine needles, revealed that the conidia were ellipsoidal in shape, initially hyaline, unicellular (Fig. 2D), becoming dark brown, and developing a thick wall, with central septum, and longitudinal striations with age (Fig. 2E). Mature conidia were 26.3±0.4 to 31.2±0.2 μm long and 11.3± 0.4 to 16±0.7 μm wide (n=60). These morphological characteristics matched previous descriptions of L. pseudotheobromae (Alves et al., 2008). For the molecular identification, the ITS and TEF-1αsequences generated in
this
study
were
deposited
in
GenBank
with
KU180477
and
KU180478,respectively.Polymerase chain reaction (PCR) of the isolates resulted in amplicons of approximately 542 bp for the ITS and 301 bp for the TEF-1αgene regions. Multiple sequence alignment of the LPS-1 sequence with those from other fungi (Fig. 3) revealed that the LPS-1 sequence exhibited 100% sequence identity to a sequence from L. pseudotheobromae, but different from the type strains of L. pseudotheobromae (CBS 116459 and CBS 374.54) by way of nucleotide difference in the TEF-1αgene region. 3.4. Optimal culture conditions for promoting antifungal activity of LPS-1 The most effective medium for promoting antifungal activity of LPS-1 was PDB. No observably difference of Bgt inhibition occurred when LPS-1 was prepared in static PDB and on a rotary shaker (85.1% and 83.8% inhibition, respectively).The static culture was chosen for the following study with the purpose of energy saving.
Many other culture
conditions had significant impact on the antifungal activity of LPS-1 (Fig. 5). Increasing the inoculum load from three mycelial plugs to nine significantly reduced antifungal activity (Fig. 5A), while pH had little impact on Bgt inhibition (Fig. 5B). The optimal temperature for Bgt inhibition was 30°C, with higher or lower temperatures significantly reducing antifungal activity (Fig. 5C). It was also found that the period of incubation significantly affected the inhibition of Bgt, with peak activity occurring at day 6 (Fig. 5D). Taken together these results indicate that the optimal conditions for maximizing the antifungal activity of LPS-1 were to inoculate PDB (pH 7) with three mycelial plugs and to incubate static cultures at 30°C for 6 days. 4. Discussion
Endophytic fungi represent a large portion of fungal species and are ubiquitous in the nature, residing in the intercellular or intracellular spaces of plant tissues for at least a portion of their life cycles without causing apparent symptoms of infection (Petrini et al., 1991). We have obtained and characterized 208 isolates belonging to 17 taxonomic groups of endophytic fungi from 26 Chinese medicinal plants. Most of the isolates (approximately 60%) belong to five taxonomic groups: Phomopsis, Acremonium, Phoma, Alternaria and Colletotrichum. However, many of the isolates recovered from the different plants appeared to have very similar morphology indicating that the same species of endophyte had been repeatedly isolated from different hosts, and that only 40 distinct morphospecies had been collected. As well as exhibiting great taxonomic diversity, some of the endophytic isolates showed a high degree of both host and tissue specificity, which is supported by other reports (Selim et al., 2011; Wiyakrutta et al., 2004; Huang et al., 2008). A large proportion of the isolates collected could not be accurately identified, a common problem with endophytic fungi, and were grouped together within the mycelia sterilia. These isolates were the most abundant accounting for 29.8% of the 208 isolates collected, and found in most of the host plants investigated. The ascomycete fungus L. pseudotheobromae has recently been differentiated from L. theobromae [anamorph: Botryosphaeria rhodina (Berk. & M.A. Curtis) Arx,] based on the differences in its conidial morphology as well as phylogenetic analyses (Alves et al., 2008). Although L. pseudotheobromae is known to be a common pathogen of pantropical trees infecting at least five species in southern China alone, including Acacia confuse, Albizia falcataria, Eucalyptus sp., Mangifera sylvatica and Paulownia fortunei (Zhao et al., 2010), it
has also recently been observed to exhibit an endophytic lifestyle being isolated from the symptomless healthy plant tissues of Camptotheca acuminate (Davidiaceae) (Wei et al., 2014) and llligera rhodantha (Hernandiaceae) (Lu et al., 2 014). To the best of our knowledge, this is the first report of L. pseudotheobromae being isolated from the stem tissues of the medicinal plant Ilex cornuta. The present research was aimed to reduce using chemicals in agriculture process and find out the most suitable non-chemical method to protect wheats against powdery mildew disease. In this study, data showed that LPS-1 culture filtrates significantly reduced disease incidence and severity of wheat powdery mildew. These results might be due to that LPS-1 inhibits disease by producing some antifungal substances. These results are in accordance with many reports that many endophytic fungi produce compounds with antifungal activity (Kusari and Spiteller, 2012; Campos et al., 2008). Endophytic fungi provide an opportunity for the discovery and production of biologically active secondary metabolites. Furthermore, they have considerable advantages over plant sources as manipulation of the fermentation conditions, by altering the medium type and culture conditions (pH, temperature and harvest point), can increase production (Kusari et al., 2012). Investigation of different culture conditions including media, agitation, inoculum load, pH and temperature as well as the duration of incubation revealed considerable variation in the antagonistic activity of LPS-1 against Bgt. The optimal conditions resulted from inoculating PDB (pH 7.0) and incubation at 30°C for 6 days without shaking. Under these conditions LPS-1 exhibited better antifungal activity in both the detached leaf and seedling assays than a 10 μg/ml application of the commercial fungicide triadimefon (Table 3), which suggests that LPS-1 has the potential to
be an excellent antimicrobial agent for the control of wheat powdery mildew. Having established the optimal fermentation conditions, further research is required to identify the bioactive compound(s) responsible for the antifungal activity of LPS-1. Endophytic fungi and their host plants coevolved over a long period of time; therefore, many plants that produce bioactive products have associations with endophytic fungi capable of producing the same compounds, perhaps as a consequence of their adaption to such a specific environment (Luiz et al., 2011). For example, the famed anticancer diterpenoid paclitaxel, which occurs in all yew species (Taxus sp.) (Suffness and Wall, 1995), is also produced by the fungus Taxomyces andreanae Strobel, Stierle & Hess, which has been isolated from the pacific yew Taxus brevifolia Nutt. (Strobel et al., 1993). The medicinal plant I. cornuta also appears to be a promising source of endophytic microorganisms capable of producing metabolites with proven antimicrobial activity (Wu et al., 2014). It is therefore interesting to note that recent studies have shown that L. pseudotheobromae can produce many bioactive compounds with medicinal properties (Wei, et al., 2014; Lu, et al., 2014). For example, it was found that L. pseudotheobromae isolate F2, which was isolated from Camptotheca
acuminate
(Davidiaceae),
produced
six
new
sulfur-containing
diketopiperazines named Lasiodipline 1-6, and that Lasiodipline 5 was confirmed to be a novel antiprotozoal compound with potency comparable to the commercial drug tinidazole (Wei et al., 2014). Similar investigation of L. pseudotheobromae isolate XSZ-3, which was isolated from the branches of Camptotheca acuminate, was found to produce four different palmarumycins, one of which was characterized as a novel anticancer compound palmarumycin LP1, which exhibited significant inhibitory activity against human
promyelocytic leukemia cells with an IC50 value of 2.39 μM. (Lu et al., 2014). These results confirm that L. pseudotheobromae has great potential as a source of novel compounds for the pharmaceutical industry. Considering that other isolates such as F2 and XSZ-3 have yielded a range of bioactive compounds it is likely that isolate LPS-1, which was isolated from the medicinal plant I. cornuta, could also be a valuable resource, and the results of the current study suggest that these may not be restricted to clinically important compounds but also agriculturally important ones.In conclusion, various endophytic fungi were harbored in traditional Chinese medicinal plants, and some of them were strong antagonists. Their representative isolates exhibited significant antifungal activities against Bgt. To the best of our knowledge this represents the first report of L. pseudotheobromae having antifungal activity against plant pathogens. Isolate LPS-1 can be potential biocontrol agent, and therefore has been selected for further research in an ongoing project.
Acknowledgments This research was supported by the MATS grant from the Department of Agriculture (CARS-03-04B), funded by the R&D Special Fund for Public Welfare Industry (Agriculture) of China (Grant Nos.201303016, 201303025), the Hubei Province Science and Technology Innovation Center (2011-620-003-3) and the Foundation for Youths of Hubei Academy of Agricultural Sciences (2015nkyjj13).
References Alves, A., Crous, P.W., Correia, A., Phillips, A.J.L., 2008. Morphological and molecular data reveal cryptic speciation in Lasiodiplodia theobromae. Fungal Divers. 31, 1-13. Aly, A.H., Debbab, A., Kjer, J., Proksch, P., 2010. Fungal endophytes from higher plants: a prolific source of phytochemicals and other bioactive natural products. Fungal Divers. 41, 1–16. Backman, P.A., Sikora, R.A., 2008. Endophytes: an emerging tool for biological control. Biol. Control 46, 1–3. Campos, F., Rosa, L., Cota, B., Caligiorne, R., Rabello, A., Alves, T., Rosa, C., Zani, C., 2008. Leishmanicidal metabolites from Cochliobolus sp., an endophytic fungus isolated from Piptadenia adiantoides (Fabaceae). PLoS Negl Trop Dis. 2, e348. Carbone, I., Kohn, L.M., 1999. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia. 91:553–556. Crous, P.W., Slippers, B., Wingfield, M.J., Rheeder, J., Marasas, W.F.O., Philips, A.J.L., Alves, A., Burgess, T., Barber, P., and Groenewald, J.Z., 2006. Phylogenetic lineages in the Botryosphaeriaceae. Stud Mycol. 55, 235–253. Curtis,F.d.,Cicco.V.D.,lima,G.,2012.Efficacy of biocontrol yeasts combined with calcium silicate or sulphur for controlling durum wheat powdery mildew and increasing grain yield components. Field Crops Res. 134, 36-46. Elad,Y., Malathrakis,N. E., Dik,A. J., 1996. Biological control of botrytis-incited diseases and powdery mildews in greenhouse crops. Crop Protect. 15, 229–240. Gao, X.N, Gong Y.F., Huo, Y.X., Han,Q.M., Kang, Z.S., Huang,L.L.,2015 Endophytic bacillus subtilis strain E1R-J is a promising biocontrol agent for wheat powdery mildew. BioMed Res.Int.2015,
462645-462652. Gong S.J., Yang L.J.,Xiang L.B., Zeng F.S., Zhang X.J., Xue M.F.,Wang H.,Shi W.Q., Yu D.Z. 2013 Baseline sensitivity of Blumeria graminis f．sp．tritic to metrafenone，quinoxyfen and bioactivitives of these fungicides. Chin. J Pestici .Sci. 15,511-515. Hijri, M., Redecker, D., Jean, A., Petetot, M.C., Voigt, K., Wöstemeye, R.J., Sanders, I.R., 2002. Identification and isolation of two ascomycete fungi from spores of the arbuscular mycorrhizal fungus Scutellospora castanea. Appl. Environ. Microbiol. 68, 4567–4573. Hallmann, J., Berg, G., Schulz, B., 2006. Isolation procedures for endophytic microorganisms. In: Schulz, B.J.E., Boyle, C.J.C., Sieber, T.N. (Eds.), Microbial Root Endophytes. Springer, Berlin-Heidelberg, pp. 299–319. Huang, W.Y., Cai, Y.Z., Hyde, K.D.,Corke, H., Sun, M., 2008. Biodiversity of endophytic fungi associated with 29 traditional Chinese medicinal plants. Fungal Divers. 33, 61-75. Ismail, A.M., Cirvilleri, G., Polizzi, G., Crous, P.W., Groenewald, J.Z., Lombard, L., 2012. Lasiodiplodia species associated with dieback disease of mango (Mangifera indica) in Egypt. Austral. Plant Pathol. 41, 649-660. Ji, H.F., Li, X.J., Zhang, H.Y., 2009. Natural products and drug discovery. Can thousands of years of ancient medical knowledge lead us to new and powerful drug combinations in the fight against cancer and dementia? EMBO Rep. 10, 194–200. Kharwar, R.N., Mishra, A., Gond, S.K., Stierle, A., Stierle, D. 2011. Anticancer compounds derived from fungal endophytes: their importance and future challenges. Nat. Prod. Rep. 28, 1208–1228.
Kiss, L.,2003. A review of fungal antagonists of powdery mildews and their potential as biocontrol agents. Pest Manage Sci.59, 475–483.
Kusari, S., Spiteller, M., 2012. Metabolomics of endophytic fungi producing associated plant secondary metabolites: progress, challenges and opportunities. In Metabolomics, U. Roessner, ed. (Rijeka, Croatia: In Tech), pp. 241–266. Kumar, S., Kaushik, N.,2013, Endophytic fungi isolated from oil-seed crop jatropha curcas produces oil and exhibit antifungal activity. PLoS One 8,e56202. Kusari, S., Hertweck, C., Spiteller, M., 2012, Chemical ecology of endophytic fungi: origins of secondary metabolites. Chem Biol. 27, 792-798. Liu W C, Shao Z R. 1994. The epidemic tendency of wheat powdery mildew in China. In: Li G B, ed., Prospects of
Plant
China, Beijing.
Protection
Research
in
China.
Science
and
Technology
Press
of
pp.135-141. (in Chinese)
Lu, X.J., ChenG., Li, Z.L., Zhang, Y.H., Wang, Z.Y., Rong, W.W., Pei, Y.H., Pan, H.Q., Hua, H.M., Bai J., 2014. Palmarumycins from the Endophytic Fungus Lasiodiplodia pseudotheobromae XSZ-3. Helvetica. Chimica. Acta. 97, 1289-1294. Luiz, H. R., Mariana, L.A., Vieira, B.B., Cota, S.J., T nia, M.A., Alves, Clos L.Z., Carlos A.R., 2011. Endophytic Fungi of Tropical Forests: A Promising Source of Bioactive Prototype Molecules for the Treatment of Neglected Diseases, Drug Development -A Case Study Based Insight into Modern Strategies, Rundfeldt, C., (Ed.), In Tech, Rijeka. pp.469-486. Petrini, O., Fungal endophytes of tree leaves. 1991. In: Andrews, N.J., Hirano, S.S., (eds), Springer, New York. pp. 179–197. Selim K.A., El-Beih, A.A., AbdEl-Rahman, T.M., El-Diwany, A.I., 2011. Biodiversity and antimicrobial activity of endophytes associated with Egyptian medicinal plants. Mycosphere 6, 669-678. Staniek, A., Woerdenbag, H.J., Kayser, O., 2008. Endophytes: exploiting biodiversity for the improvement
of natural product-based drug discovery. J. Plant Interact. 3, 75–93. Strobel, G. A., Li, J.Y., Sugawara, F., Koshino, H., Harper, J., Hess., W.M., 1999. Oocydin A, a chlorinated macrocyclic lactone with potent anti-oomycete activity from Serratia marcescens. Microbiol. 145, 3557–3564. Strobel, G. A., Stierle, A., Stierle, D., Hess, W.M., 1993. Taxomyces andreanae, a proposed new taxon for a bulbilliferous hyphomycete associated with Pacific yew (Taxus brevifolia). Mycotaxon. 47, 71–78. Strobel, G.A., Daisy, B., Castillo, U., Harper, J., 2004. Natural products from endophytic microorganisms. J. Nat. Prod. 67, 257–268. Suffness, M., Wall, M.E., 1995. Discovery and development of taxol, In Taxol: science and applications. Suffness, M. (Ed.), Taxol: Science and Applications. CRC Press, Boca Raton, Florida. pp. 1–25 Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596-1599. Vieira, M.L., Johann, S., Hughes, F.M., Rosa, C.A., Rosa, L.H., 2014. The diversity and antimicrobial activity of endophytic fungi associated with medicinal plant Baccharis trimera (Asteraceae) from the Brazilian savannah. Can. J. Microbiol. 60, 847-856. Vilgalys, R., Hester, M., 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J. Bacteriol. 172, 3238-4276. White, T.J., Bruns, T, Lee, S., Taylor, J.W., 1990. Sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J., (eds.) PCR Protocols: a guide to methods and applications. Academic Press, Inc., New York. pp. 315-322 Winter,J., M. , Behnken,S., Hertweck,C., 2011. Genomics-inspired discovery of natural products. Current Opinion in Chemical Biology, 15,22–31
Wiyakrutta, S., Sriubolmas, N., Panphut, W., Thongon, N., Danwiserkanjana, K., Ruangrungsi, N., Meevootisom, V., 2004. Endophytic fungi with antimicrobial, anti-cancer, anti-malarial activities isolated from Thai medicinal plants. World. J. Microb. Biot. 20, 265-272. Wei, W., Jiang, N., Mei, Y.N., Chu, Y.L., Ge, H.M., Song, Y.C., Ng, S.W., Tan, R.X., 2014. An antibacterial metabolite from Lasiodiplodia pseudotheobromae F2. Phytochemistry 100,103-109 Wu, Y., Zhang, X.C., Sun, J.B., Wu, X., Li, Y.Y., Gu, W., Wang, D.Y., Liu, J.G., Hu, Y.L., 2014. Chemical constituents from the leaves of Ilex urceolatus, Biochem. Syst. Ecol. 54, 92-95. Yang L., Zhou Y. L., Duan X. Y., Cao X. R., Zou Y. F., 2011.Sensitivity of Blumeria graminis f. sp. tritici isolates to triadimenfor and fenpropidin in China. Acta Phytopathol Sinica, 43, 430–434.Yang, X.J., Yang, L.J., Wang, S.N., Yu, D.Z., Ni, H.W., 2008. Effect of physcion, a natural anthraquinone derivative, on the infection process of Blumeria graminis on wheat. Can. J. Plant Pathol. 30, 391-396. Zhang, J., Wu, M.D., Li, G.Q., Yang, L., Yu, L., Jiang, D.H., Huang, H.C., Zhuang, W.Y., 2010. Botrytis fabiopsis, a new species causing chocolate spot of broad bean in central China. Mycologia. 102, 1114–1126. Zhang, Q.H., Zhang, J., Yang, L., Zhang, L., Jiang, D.H., Chen., W.D., Li, G.Q.,2014.Diversity and biocontrol potential of endophytic fungi in Brassica napus. Biol. Control 72, 98-108. Zhao, J.P., Lu, Q., Liang, J., Decock, C., Zhang, X.Y., 2010. Lasiodiplodia pseudotheobromae, a new record of pathogenic fungus from some subtropical and tropical trees in southern China. Cryptogamie Mycol. 31, 431-439.
Fig. 1. Effect of LPS-1 on the proliferation of Blumeria graminis f.sp. tritici. A, Detached leaf assay; B, Wheat seedling assay.
Fig. 2. Colony and conidial morphology of LPS-1. A, Colony morphology after 3 days incubation at 25°C; B, Colony morphology after 10 days at 25°C; C, Conidiogenous cells and young conidia; D, Hyaline, aseptate conidia. E, Septate, dark-walled conidia. The conidia micrographs were taken at ×100 magnification (oil immersion) from pycnidia formed on pine needles. Scale bar = 10 µm.
Fig. 3. Phylogenetic tree based on maximum-likelihood analysis of combined ITS and TEF-1α sequence data. Bootstrap
values >51% are presented above branches, and bootstrap values <51% are not shown. Guignardia philoprina (CBS447.68) represents the out-group.
Fig. 4. Effect of different media on the antifungal activity of LPS-1. Liquid cultures (pH 7.0) were inoculated with three mycelial plugs (5 mm in diameter) and incubated at 30℃ for 6 days: PDB, potato dextrose broth; MPDB, modified potato dextrose broth; MEB, malt extract broth; and CDB, Czapek-Dox broth.
Fig. 5. Effect of inoculum load, pH, temperature and duration of incubation on the antifungal activity of LPS-1.
Table 1 Number of endophytic fungi belonging to different taxa isolated from 26 Chinese medicinal plants Plant host\Fungal taxa
a
C
A
Ph
P
F
A
M
C
B
G
X
P
C
P
Ph
R
S
Tota
o
l
o
h
u
c
s
h
o
u
y
l
l
e
y
h
c
l
Eomecon chionantha Xylosma racemosum
1 3
1 4
Fraxinus hupehensis
5 1
1
4
Silybum marianum
marginatum Euonymus alatus
Epimedium sagitlatum
3
2
2
Buddleja lindleyana
3
4
5
3
3
3
1
1
1
10
1
4
12
1
8 2
1
4
7 1
4
2
1
Podocarpus
1
3
Nandira domestica
2
3
2
3 2
1
Narcissus
2
pseudonarcissus Syringa tomentella
16 2
6
3
3
3
5
5
16
5
9
2
5
3
8
2
3
9
1
1
16
2
Polygonum
1 1
capitatum Pinus massoniana
2 16
1 9
1 39
3 2 2
6
3
4
2
Abelia macrotera
1
4 3
2
macrophyllus
Total
13 1
1
bachli
macrolepis
8
4
2
Rhododendron
Calocedrus
1
2
Torreya grandis
chindensensevar
3
2
Papaver rhoeas
Lorpetalum
10
7
2 5
1
1
4
4
Buxus ichangensis
Peristrophe japonic
1
4
2
3
Bupleurum
Ilex cornuta
2
1
1
Buxus bodiniero
14 3
3 1
1
2
Callicarpa bodinieri Saxifraga stdonifera
1
3
2
3
3
3
12
24
62
4
4
1
3
2
1
1
1
1
2
208
a Morphologically identified fungal taxa: Fu: Fusarium spp.; Al: Alternaria spp.; Ph: Phoma spp.; Pho: Phomopsis spp.; Co: Colletotrichum spp.; Pl: Pleosporales spp.; MS: mycelia sterilia spp.; Ch: Chaetomium spp.; Bo: Botryosphaeria spp.; Gu: Guignardia spp.; Xy: Xylariales spp.; Ac: Acremonium spp.; Cl: Cladosporium spp.; Pe: Penicillium spp.; Phy: Physalospora spp.; Rh: Rhizosphaera spp.; SC: Scopulariopsis spp.;
Table 2 Number of endophytic fungi isolated from the stems, leaves and flowers of 26 Chinese medicinal plants growing in the Wuhan botanical gardens at the Chinese Academy of Sciences.
Number of fungal isolates
Plant source
Stem
Leaf
Flower
Total
Eomecon chionantha
NIa
1
NI
1
Xylosma racemosum
6
8
NI
14
Fraxinus hupehensis
NI
3
NI
3
Callicarpa bodinieri
4
NI
NI
4
Saxifraga stdonifera
7
2
NI
9
Silybum marianum
3
NI
NI
3
Buxus bodiniero
NI
7
NI
7
Bupleurum marginatum
8
NI
NI
8
Euonymus alatus
NI
13
NI
13
Ilex cornuta
6
NI
4
10
Epimedium sagitlatum
5
6
NI
11
Buddleja lindleyana
5
6
NI
11
Buxus ichangensis
NI
2
NI
2
Papaver rhoeas
NI
7
NI
7
Torreya grandis
NI
1
NI
1
Peristrophe japonic
NI
17
NI
17
Rhododendron bachli
1
5
NI
6
Podocarpus macrophyllus
8
1
NI
9
Lorpetalum chindensensevar
6
9
NI
15
Calocedrus macrolepis
7
11
NI
18
Nandira domestica
2
7
NI
9
Abelia macrotera
2
3
NI
5
Narcissus pseudonarcissus
6
2
NI
8
Syringa tomentella
NI
2
NI
2
Polygonum capitatum
1
2
NI
3
Pinus massoniana
6
6
NI
12
Total
83
121
4
208
NI = not isolated.
Table 3 Antifungal activity of selected isolates of endophytic fungi against Blumeria graminis f.sp. tritici on detached leaves and seedlings of wheat.
Detached leaves assay Treatment
Whole seedling assay
Severity1
Efficacy2
Severity
Efficacy
ESL-3
18.1±5.9efg
80.2±5.8
34.6±5.0h
57.1±5.9
LPS-1
0.0i
100
14.0±3.6i
85.0±4.9
16-6
8.7±3.2h
90.4±3.7
32.5±3.5h
59.6±5.8
PBP-4
30±4.4bc
67.0±4.2
45.7±3.4g
43.2±6.1
LPS-4
24.4±4.1bcde
73.2±3.5
52.8±7.1efg
33.0±8.8
16-3
14.4±6.0fgh
84.2±6.3
55.7±6.3def
31.1±6.4
SCS-4
26.4±4.8bcd
70.9±5.3
62.7±3.1cde
22.1±6.6
SCS-5
25.6±7.1bcd
71.6±8.9
69.0±3.0bc
14.5±1.7
SCS-6
7.9±1.0h
91.3±0.8
30.8±2.3h
63.5±2.7
EML-3
11.4±2.1gh
87.4±2.5
56.3±9.4def
30.3±9.6
BDS-3
22.5±1.7cde
75.2±2.8
70.7±5.1bc
12.4±4.8
LPF-4
29.7±6.3bc
67.4±6.1
58.8±5.9def
27.1±6.9
LKL-8
31.4±2.1b
65.4±1.9
74.1±5.0ab
8.2±2.9
PLS-1
29.7±3.4bc
67.1±4.9
64.7±6.9cd
19.9±6.9
PLS-3
17.2±1.7efg
81.0±2.0
51.3±5.5fg
36.2±8.6
CK2
19.7±2.1def
78.2±3.2
27.5±2.8h
65.8±4.7
CK1
90.8±3.8a
CK1, Negative control treated with PDB and inoculated with Bgt.
80.7±3.3a
CK2, Positive control treated with 10 μg/ml triadimefon and inoculated with Bgt. 1
Means followed by the same letters within a column do not differ significantly (P>0.05) according to Duncan’s Multiple
Range Test. 2
The biocontrol efficacy was calculated using the following formula: biocontrol efficacy (%) = (negative control
treated−treated)/ negative control treated×100%. The disease severity was investigated 12 days after the inoculation of Blumeria graminis f. sp. tritici.
Highlights
208 endophytic fungal isolates were collected from stems (83), leaves (121) and flowers (4) of 26 medicinal plants.
Fifteen endophytic fungi exhibited antifungal activity.
Strain of L. pseudotheobromae has strong biological control of wheat powdery mildew.
The first report of L. pseudotheobromae having antifungal activity against plant pathogens.
Graphical abstract