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Leaf chemistry and co-occurring species interactions affecting the endophytic fungal composition of Eupatorium adenophorum

Leaf chemistry and co-occurring species interactions affecting the endophytic fungal composition... Ann Microbiol (2011) 61:655–662 DOI 10.1007/s13213-010-0186-1 ORIGINAL ARTICLE Leaf chemistry and co-occurring species interactions affecting the endophytic fungal composition of Eupatorium adenophorum Huan Jiang & Yun-Tao Shi & Zhen-Xin Zhou & Chen Yang & Yun-Jiao Chen & Li-Min Chen & Ming-Zhi Yang & Han-Bo Zhang Received: 8 July 2010 /Accepted: 13 December 2010 /Published online: 10 January 2011 Springer-Verlag and the University of Milan 2011 Abstract The endophytic fungal composition of healthy Introduction leaves from an invasive plant (Eupatorium adenophorum)in China was investigated. Six morphologically different Foliar endophytic fungi commonly colonize and live within endophytes were found to inhabit the leaves. Based on a healthy leaves and are known to cause asymptomatic phylogenetic analysis of the internal transcribed spacer infections of the vast majority of terrestrial plants (Arnold sequences (ITS), four morphotypes were close to Alternaria, 2007). These endophytes comprise a wide variety of Cladosporium, Pestalotiopsis,and Didymella,respectively, species spanning at least four phyla of fungi (Arnold and while two morphotypes were close to unidentified fungi. The Lutzoni 2007). Most studies have focused on vertically frequency at which the endophytes occurred varied among transmitted endophytes (VTEs; mainly as Neotyphodium) leaves, branches, and individuals, with species diversity that inhabit temperate grasses (Lolium) (Saikkonen et al. markedly increasing with increasing leaf age. Chemical 1998; Müller and Krauss 2005). Conversely, the effects of conditions alone did not adequately explain the lower degree horizontally transmitted endophytes (HTEs) on their host of endophytic fungal diversity in younger leaves. Competi- plants have received little attention, despite the common tion between endophytes mediated through non-volatile and presence of these fungi in aerial tissues of a wide variety of volatile metabolites were common and may be a major factor plants (Arnold 2007). Several recent studies have shown accounting for the heterogeneous distribution of endophytes that plants can respond to HTE infections in ecologically in leaves of E. adenophorum. meaningful ways. Redman et al. (2002) demonstrated that HTEs can enhance thermotolerance of temperate plants, . . Keywords Eupatorium adenophorum Invasive plant and Arnold et al. (2003) reported that the inoculation of . . Foliar endophytes Interaction Leaf chemistry endophyte-free leaves with HTEs isolated from naturally infected asymptomatic hosts significantly decreased leaf necrosis and leaf mortality when Theobroma cacao seed- : : : : : H. Jiang Y.-T. Shi Z.-X. Zhou C. Yang Y.-J. Chen lings were challenged with a major pathogen (Phytophthora H.-B. Zhang sp.). This type of protection may be mediated by direct Laboratory of Conservation and Utilization for Bio-resources and Key interactions between endophytes and foliar pathogens. Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, In general, HTEs can accumulate on leaves shortly after Kunming, Yunnan 650091, People’s Republic of China emergence (Arnold et al. 2003), and infection densities in living leaves increase quickly after budbreak to levels that : : : : : H. Jiang Y.-T. Shi Z.-X. Zhou C. Yang Y.-J. Chen : : remain relatively constant throughout the leaf’s lifetime L.-M. Chen M.-Z. Yang H.-B. Zhang (*) School of Life Science, Yunnan University, (Arnold and Lutzoni 2007). Over time, endophyte-free leaf Kunming, Yunnan 650091, People’s Republic of China tissues become saturated with endophytes (Herre et al. e-mail: zhhb@ynu.edu.cn 2007). These changes in species composition and diversity of HTEs with increasing leaf age may be related to leaf H.-B. Zhang chemistry as the prevalence of antifungal secondary e-mail: zhbdm@yahoo.com.cn 656 Ann Microbiol (2011) 61:655–662 compounds in young leaves has been documented in many sion in a 70% (v/v) ethanol solution for 15 s, followed by tree species in tropical forests. Consequently, the lower immersion in 0.5% (w/v) sodium hypochorite solution for level of fungal diversity in young leaves may be attributed 2 min) and then placed on sterile paper in a laminar flow to strong inhibition based on leaf chemistry (Arnold and unit for 15 min to dry. The leaves were then placed on a Herre 2003). Alternatively, competitive interactions among suspended plastic mesh enclosed in sterilized plastic boxes co-occurring endophytes are likely the underlying factors to which deionized H O (25 ml) was added underneath the determining endophytic fungal distribution in an individual mesh to maintain humidity. The boxes were stored at –20°C leaf (Arnold and Lutzoni 2007). However, such interactions for 24 h to freeze plant tissue and then incubated at ambient remain to be evaluated. temperature under interval lighting for 6–8 days. The fungal Crofton weed Eupatorium adenophorum (also called colonies that erupted from the treated leaf tissue were Ageratina adenophora; family Asteraceae) is one of the counted, and representative ones were randomly selected to most invasive weeds in China. This plant, which is native inoculate onto potato dextrose agar (PDA) containing to Central America, was first recorded in Yunnan province aminobenzylpenicillin (20 μg/mL) and streptomycin sulfate in the 1940s. Following an outbreak in 1960, it has been (40 μg/mL). Pure cultures were obtained by several rounds spreading north- and eastwards at an annual rate of of single-spore streaking on new PDA. approximately 20 km through Guizhou, Sichuan, and Species density was determined as the number of each Guangxi provinces in Southwest China (Wang and Wang colony type per unit of leaf area. 2006). This weed is currently distributed in more than 30 countries, including China (Qiang 1998; Lu et al. 2005; Molecular identification of endophytes Ding et al. 2008). In an earlier unpublished study, we found a variety of endophytic fungi inhabiting the leaves rather Total genomic DNA of each endophyte was isolated using than seeds of E. adenophorum (unpublished data), leading the CTAB method (Zolan and Pukkila 1986). The rRNA us to conclude that these endophytes were acquired through genes of the internal transcribed spacer (ITS) were amplified horizontally transmission in its invaded range. However, to with ITS4 and ITS5 primers as described by White et al. date, there has been no report on leaf endophytes in this (1990). PCR products were purified and commercially exotic plant. In the study reported here, we investigated the sequenced by BGI-SEQUENCING (Shenzhen, China). The endophytic fungal composition of healthy leaves from E. obtained sequences were further edited by deleting primer adenophorum and assessed the diversity of endophytes on sequences using Editseq and Seqman software in the leaves of different ages. We also evaluated the effects of DNASTAR package (DNASTAR, Madison, WI). The leaf chemistry on the growth of endophytes and interactions phylogenetic position was characterized by the closest between endophytes that coexist in leaves. relatives, which were found by sequence blasting in the NCBI database. Phylogenetic trees were constructed using the PHYLIP package (Felsenstein 1989)with neighbor- Materials and methods joining method. Leaves collection The effect of host leaf chemistry The sample collection site was located at 24°58.292 N and The effects of host leaf chemistry on the growth of 102°37.751 E at Xishan Hill, Kunming, China. This site is endophytes were tested using the method described by dominated by E. adenophorum with a population coverage Arnold et al. (2003). Fresh and healthy leaves of different of >75%. The plant generally has eight pairs of leaves in ages were collected from wild plants, washed in tap water, the growing season (June–October). We collected the top and rinsed with deionized water (10% w/v) overnight. Leaf six pairs of leaves that had not been damaged by herbivores extracts were incorporated into water agar prior to or pathogens from three randomly selected branches of autoclaving (final concentration 10% v/v), and cylindrical three individual plants. A total of 108 leaves were collected, plugs of hyphae and agar (diameter 5 mm) were subse- stored in separate envelopes, and immediately brought back quently cultivated on test media. Colony diameters after to the laboratory. 72 h of culture at 28°C were measured. Each strain was cultured in triplicate. Endophyte isolation Non-volatile metabolite interactions among endophytes Fungal endophytes were obtained using the overnight freeze incubation technique (ONFIT; Pryor and Michailides Initially, mycelium agar plugs (6 mm) of each tested strain 2002). Briefly, the leaves were surface-sterilized (immer- were removed from the edge of a young culture and Ann Microbiol (2011) 61:655–662 657 transferred to the center of petri dishes (diameter 90 mm; ) C; or T/C − 1, when T < C) according to the method of containing PDA; a sterilized cellophane disc was also Williamson and Richardson (1988). The parameter “C” placed on the surface of the PDA. Both the culture and represents the control response and “T” represents the cellophane were removed after a 72-h culture at 28°C. An treatment response. RI>0 indicates an enhancing effect, agar plug of the target endophyte was then placed on the while RI<0 indicates an inhibition effect. The effects of all same medium and incubated at 28°C for 72 h to treatments were measured and compared by one-way demonstrate the effect of non-volatile metabolites from analysis of variance (ANOVA) using the SPSS ver. 13.0 one species against another. The control consisted of software package (SPSS, Chicago, IL). placing the target endophyte on PDA medium where previously there had been a cellophane disc but no test Nucleotide sequence accession numbers strain (Dennis and Webster 1971a). The nucleotide sequences reported in this study have been Volatile metabolite interactions among endophytes deposited in GenBank under accession numbers HM068371 to HM068376. Two petri dishes containing PDA were individually inocu- lated with a plug agar of the test and target strain, respectively. The bottoms of the plates were adjusted and Results attached by tape. The control did not contain the test strain. The cultures were incubated at 28°C for 72 h to Molecular identification of strains demonstrate the effect of volatile metabolites from one species against another (Dennis and Webster 1971b). A total of six morphologically different endophytes (identified as A, B, D, H, J, and X, respectively) were Statistical analysis observed on frozen E. adenophorum leaves. Phylogenetic analysis of ITS sequences revealed that morphotypes A, B, Shannon’s index was used to characterize species diversity D, and H were close to Alternaria, Pestalotiopsis, Didy- (Spellerberg and Fedor 2003). Interaction between strains mella, and Cladosporium, respectively. However, type X was treated as the response index (RI=1 − C/T, when T ≥ and J were most similar to unidentified fungi (Fig. 1). Fig. 1 Phylogenetic Alternaria mali (AY154683) relationship of internal Alternaria longipes (AY154684) transcribed spacer (ITS) gene sequences. Strains indicated in Alternaria sp. XSH1 (type A) bold are the endophytes Alternaria alternate (FJ904919) identified in this study. The Alternaria tenuissima (EU326185) GenBank accession numbers of reference strains are shown in Didymella sp. MD1 (type D) parenthesis. Bootstrap values Didymella cucurbitacearum (AY293804) (1,000 replications) ≥50% are indicated at branch nodes. Scale Didymella bryoniae (AB266850) bar: 10% of estimated sequence Morphotype J4 (type J) divergence Uncultured fungus (FJ213550) Pestalotiopsis sp. B2 (type B) Pestalotiopsis bicilia (AF409973) Pestalotiopsis neglecta (AY682934) Pestalotiopsis vismiae (AF409977) Cladosporium tenuissimum (AJ300331) Cladosporium cladosporioides (DQ810182) Cladosporium sp. H1 (type H) Cladosporium gossypiicola (AF393702) Cladosporium coralloides (AF393695) 100 Morphotype X6 (type X) 0.1 Fungal isolate TRN43 (AY843057) 658 Ann Microbiol (2011) 61:655–662 Endophyte diversity and distribution a 100% Endophyte number varied among leaves, branches, and 80% individuals. There were significant differences in three D individuals (P<0.05) of types B and J, but not for others. J 60% Among the nine branches, types A and D were stable in number, but types B, H, J, and X showed considerable 40% variation (P<0.05; data not shown). Type X was predom- inate in young leaves, which included >90% of colonies in 20% the first and second pair of leaves, with a colony density of 2.4 and 2.8/mm of leaf area, respectively. However, this 0% density declined to 0.5/mm of leaf area for the sixth pair of 1st 2nd 3rd 4th 5th 6th leaves. In contrast, type D tended to live in old leaves, with 0.6 colonies/mm of leaf area for the sixth leaf pair; 1.4 however, 0.01 colonies/mm was recorded for the first pair 1.2 of leaves. Type H, A, B, and J mainly coexisted in the 3rd, 4th and 5th pairs of leaves (Figs. 2, 3a). Interestingly, there 0.8 was a general tendency for endophyte diversity to increase 0.6 with leaf age, although it was slightly reduced in the sixth 0.4 leaf pair (Fig. 3b). On average, 1.2 species were observed 0.2 in the first pair of leaves, which is significantly lower than that of the second pair of leaves (about 3.0 species). The 1st 2nd 3rd 4th 5th 6th highest number of species was observed in the third, fourth Pair of leaves and fifth pair of leaves, nearly 4.1 species per leaf (data not Fig. 3 Frequency of occurrence (a) and diversity index (b)of shown). endophytes observed in each pair of leaves (1–6). A, B, D, H, J, X Morphologically different endophytes identified Fig. 2 Colony density of six 0.07 0.40 isolates on differently aged Type A Type B 0.06 leaves (leaf pairs 1–6) of 0.30 0.05 Eupatorium adenophorum. 0.04 Error bar: 1 standard error of 0.20 0.03 the mean (SEM) 0.02 0.10 0.01 0.00 0.00 1st 2nd 3rd 4th 5th 6th 1st 2nd 3rd 4th 5th 6th 0.80 0.12 Type D Type H 0.10 0.60 0.08 0.40 0.06 0.04 0.20 0.02 0.00 0.00 1st 2nd 3rd 4th 5th 6th 1st 2nd 3rd 4th 5th 6th 0.10 4.00 Type J Type X 0.08 3.00 0.06 2.00 0.04 1.00 0.02 0.00 0.00 1st 2nd 3rd 4th 5th 6th 1st 2nd 3rd 4th 5th 6th Pair of leaves Colony numbers per mm of leaf area Occurrence frequency of colony Shannon index Ann Microbiol (2011) 61:655–662 659 The effect of host leaf chemistry was age dependent, with a positive trend in young leaves (the second, third, and fourth pair of leaves) but a negative Endophytes grow differently on media containing extracts trend in old leaves (the fifth and sixth pair of leaves). from six pairs of leaves (Fig. 4). The growth rates of types A and X were inhibited significantly by the leaf extracts, Non-volatile metabolite interactions among endophytes but no difference was found between different leaves. Leaf extracts also inhibited the growth of types H and J, but Most non-volatile metabolite interactions between strains young leaves (the first pair of leaves) significantly inhibited were negative (Table 1). For example, types B, H, J, and D the growth of type H. In general, the adverse effect of old were inhibited by most of the antagonists, with type D leaves (the fifth and sixth pairs of leaves) on the growth of significantly inhibited by types B, H, and J, type H inhibited type J was greater than that of young leaves. by type X, type B inhibited by type H, and type J inhibited In contrast, the growth of type D on the control media by type D. None of the tested strains significantly inhibited exceeded that on media without leaf extracts. Relatively the growth of type X. However, five species were found to older leaves were more favorable to the growth of D, with the promote the growth of type A to some degree, especially sixth pair of leaves having a particularly significant effect type H. Interactions between some types were mutual; for (P<0.05). In addition, the effect of leaf chemistry on type B example, type J inhibited D and vice versa (Table 1). Fig. 4 Effect of host leaf 5. 00 2. 30 Type H a Type A bcd chemistry on the growth of 4. 90 endophytes. Columns 2. 20 bcde 4. 80 ac ae (mean ± SE) with the same ad 4. 70 2. 10 ab letter are not significantly 4. 60 different at P<0.05 b b 4. 50 b 2. 00 a 4. 40 1. 90 4. 30 4. 20 1. 80 4. 10 4. 00 1. 70 con 1st 2nd 3rd 4th 5th 6th con 1st 2nd 3rd 4th 5th 6th 3. 80 Type J bcd 6. 10 Type B 3. 75 ab 3. 70 6. 00 ad ab abd ad 3. 65 abd 5. 90 ac 3. 60 5. 80 bd 3. 55 cd 3. 50 5. 70 3. 45 5. 60 3. 40 5. 50 3. 35 5. 40 3. 30 con 1st 2nd 3rd 4th 5th 6th con 1st 2nd 3rd 4th 5th 6th 4. 70 1. 60 dfe Type D Type X bcfe 1. 40 4. 60 1. 20 b ae b 4. 50 acd b af 1. 00 ab 4. 40 0. 80 4. 30 0. 60 4. 20 0. 40 4. 10 0. 20 0. 00 4. 00 con 1st 2nd 3rd 4th 5th 6th con 1st 2nd 3rd 4th 5th 6th Pair of leaves Diameter of colony (cm) 660 Ann Microbiol (2011) 61:655–662 Table 1 Response indexes of endophyte interactions mediated through non-volatile metabolites Test strain type Target strain AB H J D X A −0.03±0.06 −0.01±0.00 −0.10±0.05 0.00±0.00 0.05±0.05 B 0.03±0.06* −0.06±0.16 0.04±0.04 −0.18±0.15* 0.34±0.05 H 0.12±0.00 −0.14±0.00* −0.05±0.00 −0.02±0.00* −0.14±0.37 J 0.03±0.02 −0.08±0.06 −0.01±0.00 −0.31±0.14* 0.17±0.00 D 0.07±0.02 −0.08±0.03 −0.01±0.01 −0.12±0.02* 0.20±0.06 X 0.07±0.05 −0.04±0.01 −0.02±0.00* 0.00±0.05 −0.06±0.00 *Significant difference at P<0.05 Response index (RI)>0 indicates an enhancing effect; RI<0 indicates an inhibition effect Volatile metabolite interactions among endophytes which is slightly lower than those previously reported. This may be due to the ONFIT method: although ONFIT allows Similarly, most interactions between strains through vola- fungi to be grown under conditions similar to the natural tiles were also negative (Table 2). Type A was obviously condition without the interruption of a step with artificial inhibited by types B and J, type B was also restrained by medium, it may underestimate species richness due to type A, type J was suppressed by type D, and type D was possible competition among endophytes when grown on a inhibited by types A and B. No significant stimulation whole leaf. Subsequent experiments provided evidence mediated by volatile metabolites was observed between supporting this possibility as most interactions between strains. strains mediated through volatile or non-volatile metabo- lites were competitive (Tables 1, 2). In general, the species composition of HTEs in woody Discussion plants varies with habitat, while infection density and frequency tend to increase with increasing leaf age (Arnold Unlike VTEs, such as the clavicipitaceous endophytes that and Herre 2003). Indeed, differences between three indi- mainly live in grasses, HTEs have been found to associate viduals and between nine branches were observed (data not with leaves of a variety of plants. For example, in leaves of shown). However, variations between leaves of a different tropical dicotyledonous hosts, the infection density of HTEs age were more significant, and young leaves had a less often approaches 1 endophytic isolate/2 mm of leaf area. diverse fungal community than older leaves (Fig. 3). This Nearly ten species of endophytes typically inhabit individ- difference may be attributed to the leaf chemistry of young ual tropical leaves (Arnold and Herre 2003). In moist leaves of many tropical tree species in which the prevalence tropical forests, up to 17 species of endophytes have been of antifungal secondary compounds, such as simple recovered from a single leaf, with infection domains phenolics and condensed tannins, has strong inhibitory typically on the scale of only 2 mm of leaf tissue (Arnold effects and results in a lower level of fungal diversity and Lutzoni 2007). In our study, fungal infection density (Arnold and Herre 2003). varied greatly among the different endophytic species. On To evaluate the effects of E. adenophorum leaf chem- average, four species were observed in a mature leaf of E. icals on endophytes, we investigated whether the growth of adenophorum (third, fourth, and fifth leaf pairs; Fig. 3), the isolated endophytes was sensitive to leaf chemistry. In Table 2 Response indexes of Test strain Target strain endophyte interactions mediated through volatile metabolites AB H J D X A −0.10±0.04* 0.10±0.02 −0.09±0.02 −0.15±0.01* 0.17±0.10 B −0.07±0.01* 0.09±0.11 0.07±0.05 −0.22±0.03* 0.30±0.03 H 0.01±0.02 −0.07±0.02 0.04±0.02 0.01±0.02 0.27±0.10 *Significant difference at P<0.05 J −0.05±0.01* −0.06±0.02 0.03±0.03 −0.06±0.03 0.20±0.03 D −0.01±0.00 −0.05±0.02 −0.03±0.03 −0.35±0.23* 0.20±0.03 RI>0 indicates an enhancing effect; RI<0 indicates an X −0.04±0.02 −0.07±0.03 0.00±0.00 0.04±0.03 −0.08±0.04 inhibition effect Ann Microbiol (2011) 61:655–662 661 most cases, young leaves did not show a stronger inhibitory than native Q. ilex in Spain and Switzerland. Similarly, effect on the growth of endophytes than older leaves, with introduced Eucalyptus nitens in England contained a lower the exception of the first pair of leaves which had slightly diversity of fungi than native E. nitens in Australia, as well suppressed the growth of type H when compared to other as fewer host-specific fungi and more generalists (Bayman old leaves (Fig. 4). These data indicate that most endo- et al. 1998). We were unable to demonstrate whether there phytic fungi in E. adenophorum were relatively insensitive is a difference between native and naturalized E. adeno- to the chemical conditions in differently aged leaves. phorum. However, a number of previous studies have Therefore, short colonization time trends may be the shown that endophytes of E. adenophorum tend to be major reason for the low diversity of fungi in young generalists. For example, Alternaria species are mainly leaves of E. adenophorum. Based on the changes in saprophytic fungi (Thomma 2003). Akimitsu et al. (2003) fungal diversity with increasing leaf age (Fig. 3), we consistently recovered A. alternata from healthy rough conclude that endophytic fungi in E. adenophorum can lemon tissue of asymptomatic leaves and found the accumulate on leaves shortly after emergence (about isolated strains to be unrelated to isolates causing disease. 7 days) and that infection densities increase steadily, A study also indicated that the most frequent genera of reaching a constant level after nearly 1 month (i.e., the endophytes were Alternaria from invasive spotted knap- fourth pair of leaves). weed (Centaurea stoebe, Asteraceae) in its native and Within a leaf, the distribution of diverse fungal species invaded ranges (Shipunov et al. 2008). Moreover, Clado- generally resembles a quilt-like patchwork, with different sporium is also dominant in fungal endophytes associated species usually abutting each other, producing an extremely with Citrus limon (Durán et al. 2005) and in rice (Shankar heterogeneous mix of various fungal species and genotypes Naik et al. 2009). Nevertheless, some endophytes were at very fine scales within the leaf matrix (Herre et al. 2007). found to be phylogenetically distinct from identified and In our study, the use of ONFIT allowed us to observe cultured fungus in GenBank (e.g., type X). However, we directly the heterogeneous distribution of species where a cannot conclude whether these are specific to E. adeno- few of the colonies were overlapping (data not shown). We phorum. Regardless of their specificity, it is evident that also found that each species showed a preference for some HTEs facilitate the host’s resistance response to differently aged leaves, with type X dominate in young pathogenic attacks (Arnold et al. 2003). The functions of leaves, type D flourishing in old leaves, types H and type J such endophytes in providing resistance to pathogens and coexisting in the third pair of leaves, type A dominate in the herbivores may be useful for exploring the invasive fourth pair of leaves, and type B dominate in the fourth and mechanisms of E. adenophorum. fifth pair of leaves (Fig. 3a). Subsequent experiments indicated that the heterogeneous Acknowledgments We thank the anonymous reviewer for detailed and constructive suggestions for our paper. This project was distribution and changes in diversity with increasing leaf financially supported by National Basic Research Program of China age are balanced by leaf chemistry and interactions between (973 Program) (2007CB411600), National Science Foundation of coexisting species. For example, types A and X were China (No. 30960077) and the Department of Science and Technology significantly inhibited by leaf chemistry (Fig. 4); however, of Yunnan Province, China (No.2007PY01-24). most interactions between coexisting species were positive, either through the mediation of volatile or non-volatile metabolites. All species promoted the growth of type X References through non-volatile metabolites, with the exception of type H (Table 1, 2). 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Leaf chemistry and co-occurring species interactions affecting the endophytic fungal composition of Eupatorium adenophorum

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Publisher
Springer Journals
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Copyright © 2011 by Springer-Verlag and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
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1869-2044
DOI
10.1007/s13213-010-0186-1
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Ann Microbiol (2011) 61:655–662 DOI 10.1007/s13213-010-0186-1 ORIGINAL ARTICLE Leaf chemistry and co-occurring species interactions affecting the endophytic fungal composition of Eupatorium adenophorum Huan Jiang & Yun-Tao Shi & Zhen-Xin Zhou & Chen Yang & Yun-Jiao Chen & Li-Min Chen & Ming-Zhi Yang & Han-Bo Zhang Received: 8 July 2010 /Accepted: 13 December 2010 /Published online: 10 January 2011 Springer-Verlag and the University of Milan 2011 Abstract The endophytic fungal composition of healthy Introduction leaves from an invasive plant (Eupatorium adenophorum)in China was investigated. Six morphologically different Foliar endophytic fungi commonly colonize and live within endophytes were found to inhabit the leaves. Based on a healthy leaves and are known to cause asymptomatic phylogenetic analysis of the internal transcribed spacer infections of the vast majority of terrestrial plants (Arnold sequences (ITS), four morphotypes were close to Alternaria, 2007). These endophytes comprise a wide variety of Cladosporium, Pestalotiopsis,and Didymella,respectively, species spanning at least four phyla of fungi (Arnold and while two morphotypes were close to unidentified fungi. The Lutzoni 2007). Most studies have focused on vertically frequency at which the endophytes occurred varied among transmitted endophytes (VTEs; mainly as Neotyphodium) leaves, branches, and individuals, with species diversity that inhabit temperate grasses (Lolium) (Saikkonen et al. markedly increasing with increasing leaf age. Chemical 1998; Müller and Krauss 2005). Conversely, the effects of conditions alone did not adequately explain the lower degree horizontally transmitted endophytes (HTEs) on their host of endophytic fungal diversity in younger leaves. Competi- plants have received little attention, despite the common tion between endophytes mediated through non-volatile and presence of these fungi in aerial tissues of a wide variety of volatile metabolites were common and may be a major factor plants (Arnold 2007). Several recent studies have shown accounting for the heterogeneous distribution of endophytes that plants can respond to HTE infections in ecologically in leaves of E. adenophorum. meaningful ways. Redman et al. (2002) demonstrated that HTEs can enhance thermotolerance of temperate plants, . . Keywords Eupatorium adenophorum Invasive plant and Arnold et al. (2003) reported that the inoculation of . . Foliar endophytes Interaction Leaf chemistry endophyte-free leaves with HTEs isolated from naturally infected asymptomatic hosts significantly decreased leaf necrosis and leaf mortality when Theobroma cacao seed- : : : : : H. Jiang Y.-T. Shi Z.-X. Zhou C. Yang Y.-J. Chen lings were challenged with a major pathogen (Phytophthora H.-B. Zhang sp.). This type of protection may be mediated by direct Laboratory of Conservation and Utilization for Bio-resources and Key interactions between endophytes and foliar pathogens. Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, In general, HTEs can accumulate on leaves shortly after Kunming, Yunnan 650091, People’s Republic of China emergence (Arnold et al. 2003), and infection densities in living leaves increase quickly after budbreak to levels that : : : : : H. Jiang Y.-T. Shi Z.-X. Zhou C. Yang Y.-J. Chen : : remain relatively constant throughout the leaf’s lifetime L.-M. Chen M.-Z. Yang H.-B. Zhang (*) School of Life Science, Yunnan University, (Arnold and Lutzoni 2007). Over time, endophyte-free leaf Kunming, Yunnan 650091, People’s Republic of China tissues become saturated with endophytes (Herre et al. e-mail: zhhb@ynu.edu.cn 2007). These changes in species composition and diversity of HTEs with increasing leaf age may be related to leaf H.-B. Zhang chemistry as the prevalence of antifungal secondary e-mail: zhbdm@yahoo.com.cn 656 Ann Microbiol (2011) 61:655–662 compounds in young leaves has been documented in many sion in a 70% (v/v) ethanol solution for 15 s, followed by tree species in tropical forests. Consequently, the lower immersion in 0.5% (w/v) sodium hypochorite solution for level of fungal diversity in young leaves may be attributed 2 min) and then placed on sterile paper in a laminar flow to strong inhibition based on leaf chemistry (Arnold and unit for 15 min to dry. The leaves were then placed on a Herre 2003). Alternatively, competitive interactions among suspended plastic mesh enclosed in sterilized plastic boxes co-occurring endophytes are likely the underlying factors to which deionized H O (25 ml) was added underneath the determining endophytic fungal distribution in an individual mesh to maintain humidity. The boxes were stored at –20°C leaf (Arnold and Lutzoni 2007). However, such interactions for 24 h to freeze plant tissue and then incubated at ambient remain to be evaluated. temperature under interval lighting for 6–8 days. The fungal Crofton weed Eupatorium adenophorum (also called colonies that erupted from the treated leaf tissue were Ageratina adenophora; family Asteraceae) is one of the counted, and representative ones were randomly selected to most invasive weeds in China. This plant, which is native inoculate onto potato dextrose agar (PDA) containing to Central America, was first recorded in Yunnan province aminobenzylpenicillin (20 μg/mL) and streptomycin sulfate in the 1940s. Following an outbreak in 1960, it has been (40 μg/mL). Pure cultures were obtained by several rounds spreading north- and eastwards at an annual rate of of single-spore streaking on new PDA. approximately 20 km through Guizhou, Sichuan, and Species density was determined as the number of each Guangxi provinces in Southwest China (Wang and Wang colony type per unit of leaf area. 2006). This weed is currently distributed in more than 30 countries, including China (Qiang 1998; Lu et al. 2005; Molecular identification of endophytes Ding et al. 2008). In an earlier unpublished study, we found a variety of endophytic fungi inhabiting the leaves rather Total genomic DNA of each endophyte was isolated using than seeds of E. adenophorum (unpublished data), leading the CTAB method (Zolan and Pukkila 1986). The rRNA us to conclude that these endophytes were acquired through genes of the internal transcribed spacer (ITS) were amplified horizontally transmission in its invaded range. However, to with ITS4 and ITS5 primers as described by White et al. date, there has been no report on leaf endophytes in this (1990). PCR products were purified and commercially exotic plant. In the study reported here, we investigated the sequenced by BGI-SEQUENCING (Shenzhen, China). The endophytic fungal composition of healthy leaves from E. obtained sequences were further edited by deleting primer adenophorum and assessed the diversity of endophytes on sequences using Editseq and Seqman software in the leaves of different ages. We also evaluated the effects of DNASTAR package (DNASTAR, Madison, WI). The leaf chemistry on the growth of endophytes and interactions phylogenetic position was characterized by the closest between endophytes that coexist in leaves. relatives, which were found by sequence blasting in the NCBI database. Phylogenetic trees were constructed using the PHYLIP package (Felsenstein 1989)with neighbor- Materials and methods joining method. Leaves collection The effect of host leaf chemistry The sample collection site was located at 24°58.292 N and The effects of host leaf chemistry on the growth of 102°37.751 E at Xishan Hill, Kunming, China. This site is endophytes were tested using the method described by dominated by E. adenophorum with a population coverage Arnold et al. (2003). Fresh and healthy leaves of different of >75%. The plant generally has eight pairs of leaves in ages were collected from wild plants, washed in tap water, the growing season (June–October). We collected the top and rinsed with deionized water (10% w/v) overnight. Leaf six pairs of leaves that had not been damaged by herbivores extracts were incorporated into water agar prior to or pathogens from three randomly selected branches of autoclaving (final concentration 10% v/v), and cylindrical three individual plants. A total of 108 leaves were collected, plugs of hyphae and agar (diameter 5 mm) were subse- stored in separate envelopes, and immediately brought back quently cultivated on test media. Colony diameters after to the laboratory. 72 h of culture at 28°C were measured. Each strain was cultured in triplicate. Endophyte isolation Non-volatile metabolite interactions among endophytes Fungal endophytes were obtained using the overnight freeze incubation technique (ONFIT; Pryor and Michailides Initially, mycelium agar plugs (6 mm) of each tested strain 2002). Briefly, the leaves were surface-sterilized (immer- were removed from the edge of a young culture and Ann Microbiol (2011) 61:655–662 657 transferred to the center of petri dishes (diameter 90 mm; ) C; or T/C − 1, when T < C) according to the method of containing PDA; a sterilized cellophane disc was also Williamson and Richardson (1988). The parameter “C” placed on the surface of the PDA. Both the culture and represents the control response and “T” represents the cellophane were removed after a 72-h culture at 28°C. An treatment response. RI>0 indicates an enhancing effect, agar plug of the target endophyte was then placed on the while RI<0 indicates an inhibition effect. The effects of all same medium and incubated at 28°C for 72 h to treatments were measured and compared by one-way demonstrate the effect of non-volatile metabolites from analysis of variance (ANOVA) using the SPSS ver. 13.0 one species against another. The control consisted of software package (SPSS, Chicago, IL). placing the target endophyte on PDA medium where previously there had been a cellophane disc but no test Nucleotide sequence accession numbers strain (Dennis and Webster 1971a). The nucleotide sequences reported in this study have been Volatile metabolite interactions among endophytes deposited in GenBank under accession numbers HM068371 to HM068376. Two petri dishes containing PDA were individually inocu- lated with a plug agar of the test and target strain, respectively. The bottoms of the plates were adjusted and Results attached by tape. The control did not contain the test strain. The cultures were incubated at 28°C for 72 h to Molecular identification of strains demonstrate the effect of volatile metabolites from one species against another (Dennis and Webster 1971b). A total of six morphologically different endophytes (identified as A, B, D, H, J, and X, respectively) were Statistical analysis observed on frozen E. adenophorum leaves. Phylogenetic analysis of ITS sequences revealed that morphotypes A, B, Shannon’s index was used to characterize species diversity D, and H were close to Alternaria, Pestalotiopsis, Didy- (Spellerberg and Fedor 2003). Interaction between strains mella, and Cladosporium, respectively. However, type X was treated as the response index (RI=1 − C/T, when T ≥ and J were most similar to unidentified fungi (Fig. 1). Fig. 1 Phylogenetic Alternaria mali (AY154683) relationship of internal Alternaria longipes (AY154684) transcribed spacer (ITS) gene sequences. Strains indicated in Alternaria sp. XSH1 (type A) bold are the endophytes Alternaria alternate (FJ904919) identified in this study. The Alternaria tenuissima (EU326185) GenBank accession numbers of reference strains are shown in Didymella sp. MD1 (type D) parenthesis. Bootstrap values Didymella cucurbitacearum (AY293804) (1,000 replications) ≥50% are indicated at branch nodes. Scale Didymella bryoniae (AB266850) bar: 10% of estimated sequence Morphotype J4 (type J) divergence Uncultured fungus (FJ213550) Pestalotiopsis sp. B2 (type B) Pestalotiopsis bicilia (AF409973) Pestalotiopsis neglecta (AY682934) Pestalotiopsis vismiae (AF409977) Cladosporium tenuissimum (AJ300331) Cladosporium cladosporioides (DQ810182) Cladosporium sp. H1 (type H) Cladosporium gossypiicola (AF393702) Cladosporium coralloides (AF393695) 100 Morphotype X6 (type X) 0.1 Fungal isolate TRN43 (AY843057) 658 Ann Microbiol (2011) 61:655–662 Endophyte diversity and distribution a 100% Endophyte number varied among leaves, branches, and 80% individuals. There were significant differences in three D individuals (P<0.05) of types B and J, but not for others. J 60% Among the nine branches, types A and D were stable in number, but types B, H, J, and X showed considerable 40% variation (P<0.05; data not shown). Type X was predom- inate in young leaves, which included >90% of colonies in 20% the first and second pair of leaves, with a colony density of 2.4 and 2.8/mm of leaf area, respectively. However, this 0% density declined to 0.5/mm of leaf area for the sixth pair of 1st 2nd 3rd 4th 5th 6th leaves. In contrast, type D tended to live in old leaves, with 0.6 colonies/mm of leaf area for the sixth leaf pair; 1.4 however, 0.01 colonies/mm was recorded for the first pair 1.2 of leaves. Type H, A, B, and J mainly coexisted in the 3rd, 4th and 5th pairs of leaves (Figs. 2, 3a). Interestingly, there 0.8 was a general tendency for endophyte diversity to increase 0.6 with leaf age, although it was slightly reduced in the sixth 0.4 leaf pair (Fig. 3b). On average, 1.2 species were observed 0.2 in the first pair of leaves, which is significantly lower than that of the second pair of leaves (about 3.0 species). The 1st 2nd 3rd 4th 5th 6th highest number of species was observed in the third, fourth Pair of leaves and fifth pair of leaves, nearly 4.1 species per leaf (data not Fig. 3 Frequency of occurrence (a) and diversity index (b)of shown). endophytes observed in each pair of leaves (1–6). A, B, D, H, J, X Morphologically different endophytes identified Fig. 2 Colony density of six 0.07 0.40 isolates on differently aged Type A Type B 0.06 leaves (leaf pairs 1–6) of 0.30 0.05 Eupatorium adenophorum. 0.04 Error bar: 1 standard error of 0.20 0.03 the mean (SEM) 0.02 0.10 0.01 0.00 0.00 1st 2nd 3rd 4th 5th 6th 1st 2nd 3rd 4th 5th 6th 0.80 0.12 Type D Type H 0.10 0.60 0.08 0.40 0.06 0.04 0.20 0.02 0.00 0.00 1st 2nd 3rd 4th 5th 6th 1st 2nd 3rd 4th 5th 6th 0.10 4.00 Type J Type X 0.08 3.00 0.06 2.00 0.04 1.00 0.02 0.00 0.00 1st 2nd 3rd 4th 5th 6th 1st 2nd 3rd 4th 5th 6th Pair of leaves Colony numbers per mm of leaf area Occurrence frequency of colony Shannon index Ann Microbiol (2011) 61:655–662 659 The effect of host leaf chemistry was age dependent, with a positive trend in young leaves (the second, third, and fourth pair of leaves) but a negative Endophytes grow differently on media containing extracts trend in old leaves (the fifth and sixth pair of leaves). from six pairs of leaves (Fig. 4). The growth rates of types A and X were inhibited significantly by the leaf extracts, Non-volatile metabolite interactions among endophytes but no difference was found between different leaves. Leaf extracts also inhibited the growth of types H and J, but Most non-volatile metabolite interactions between strains young leaves (the first pair of leaves) significantly inhibited were negative (Table 1). For example, types B, H, J, and D the growth of type H. In general, the adverse effect of old were inhibited by most of the antagonists, with type D leaves (the fifth and sixth pairs of leaves) on the growth of significantly inhibited by types B, H, and J, type H inhibited type J was greater than that of young leaves. by type X, type B inhibited by type H, and type J inhibited In contrast, the growth of type D on the control media by type D. None of the tested strains significantly inhibited exceeded that on media without leaf extracts. Relatively the growth of type X. However, five species were found to older leaves were more favorable to the growth of D, with the promote the growth of type A to some degree, especially sixth pair of leaves having a particularly significant effect type H. Interactions between some types were mutual; for (P<0.05). In addition, the effect of leaf chemistry on type B example, type J inhibited D and vice versa (Table 1). Fig. 4 Effect of host leaf 5. 00 2. 30 Type H a Type A bcd chemistry on the growth of 4. 90 endophytes. Columns 2. 20 bcde 4. 80 ac ae (mean ± SE) with the same ad 4. 70 2. 10 ab letter are not significantly 4. 60 different at P<0.05 b b 4. 50 b 2. 00 a 4. 40 1. 90 4. 30 4. 20 1. 80 4. 10 4. 00 1. 70 con 1st 2nd 3rd 4th 5th 6th con 1st 2nd 3rd 4th 5th 6th 3. 80 Type J bcd 6. 10 Type B 3. 75 ab 3. 70 6. 00 ad ab abd ad 3. 65 abd 5. 90 ac 3. 60 5. 80 bd 3. 55 cd 3. 50 5. 70 3. 45 5. 60 3. 40 5. 50 3. 35 5. 40 3. 30 con 1st 2nd 3rd 4th 5th 6th con 1st 2nd 3rd 4th 5th 6th 4. 70 1. 60 dfe Type D Type X bcfe 1. 40 4. 60 1. 20 b ae b 4. 50 acd b af 1. 00 ab 4. 40 0. 80 4. 30 0. 60 4. 20 0. 40 4. 10 0. 20 0. 00 4. 00 con 1st 2nd 3rd 4th 5th 6th con 1st 2nd 3rd 4th 5th 6th Pair of leaves Diameter of colony (cm) 660 Ann Microbiol (2011) 61:655–662 Table 1 Response indexes of endophyte interactions mediated through non-volatile metabolites Test strain type Target strain AB H J D X A −0.03±0.06 −0.01±0.00 −0.10±0.05 0.00±0.00 0.05±0.05 B 0.03±0.06* −0.06±0.16 0.04±0.04 −0.18±0.15* 0.34±0.05 H 0.12±0.00 −0.14±0.00* −0.05±0.00 −0.02±0.00* −0.14±0.37 J 0.03±0.02 −0.08±0.06 −0.01±0.00 −0.31±0.14* 0.17±0.00 D 0.07±0.02 −0.08±0.03 −0.01±0.01 −0.12±0.02* 0.20±0.06 X 0.07±0.05 −0.04±0.01 −0.02±0.00* 0.00±0.05 −0.06±0.00 *Significant difference at P<0.05 Response index (RI)>0 indicates an enhancing effect; RI<0 indicates an inhibition effect Volatile metabolite interactions among endophytes which is slightly lower than those previously reported. This may be due to the ONFIT method: although ONFIT allows Similarly, most interactions between strains through vola- fungi to be grown under conditions similar to the natural tiles were also negative (Table 2). Type A was obviously condition without the interruption of a step with artificial inhibited by types B and J, type B was also restrained by medium, it may underestimate species richness due to type A, type J was suppressed by type D, and type D was possible competition among endophytes when grown on a inhibited by types A and B. No significant stimulation whole leaf. Subsequent experiments provided evidence mediated by volatile metabolites was observed between supporting this possibility as most interactions between strains. strains mediated through volatile or non-volatile metabo- lites were competitive (Tables 1, 2). In general, the species composition of HTEs in woody Discussion plants varies with habitat, while infection density and frequency tend to increase with increasing leaf age (Arnold Unlike VTEs, such as the clavicipitaceous endophytes that and Herre 2003). Indeed, differences between three indi- mainly live in grasses, HTEs have been found to associate viduals and between nine branches were observed (data not with leaves of a variety of plants. For example, in leaves of shown). However, variations between leaves of a different tropical dicotyledonous hosts, the infection density of HTEs age were more significant, and young leaves had a less often approaches 1 endophytic isolate/2 mm of leaf area. diverse fungal community than older leaves (Fig. 3). This Nearly ten species of endophytes typically inhabit individ- difference may be attributed to the leaf chemistry of young ual tropical leaves (Arnold and Herre 2003). In moist leaves of many tropical tree species in which the prevalence tropical forests, up to 17 species of endophytes have been of antifungal secondary compounds, such as simple recovered from a single leaf, with infection domains phenolics and condensed tannins, has strong inhibitory typically on the scale of only 2 mm of leaf tissue (Arnold effects and results in a lower level of fungal diversity and Lutzoni 2007). In our study, fungal infection density (Arnold and Herre 2003). varied greatly among the different endophytic species. On To evaluate the effects of E. adenophorum leaf chem- average, four species were observed in a mature leaf of E. icals on endophytes, we investigated whether the growth of adenophorum (third, fourth, and fifth leaf pairs; Fig. 3), the isolated endophytes was sensitive to leaf chemistry. In Table 2 Response indexes of Test strain Target strain endophyte interactions mediated through volatile metabolites AB H J D X A −0.10±0.04* 0.10±0.02 −0.09±0.02 −0.15±0.01* 0.17±0.10 B −0.07±0.01* 0.09±0.11 0.07±0.05 −0.22±0.03* 0.30±0.03 H 0.01±0.02 −0.07±0.02 0.04±0.02 0.01±0.02 0.27±0.10 *Significant difference at P<0.05 J −0.05±0.01* −0.06±0.02 0.03±0.03 −0.06±0.03 0.20±0.03 D −0.01±0.00 −0.05±0.02 −0.03±0.03 −0.35±0.23* 0.20±0.03 RI>0 indicates an enhancing effect; RI<0 indicates an X −0.04±0.02 −0.07±0.03 0.00±0.00 0.04±0.03 −0.08±0.04 inhibition effect Ann Microbiol (2011) 61:655–662 661 most cases, young leaves did not show a stronger inhibitory than native Q. ilex in Spain and Switzerland. Similarly, effect on the growth of endophytes than older leaves, with introduced Eucalyptus nitens in England contained a lower the exception of the first pair of leaves which had slightly diversity of fungi than native E. nitens in Australia, as well suppressed the growth of type H when compared to other as fewer host-specific fungi and more generalists (Bayman old leaves (Fig. 4). These data indicate that most endo- et al. 1998). We were unable to demonstrate whether there phytic fungi in E. adenophorum were relatively insensitive is a difference between native and naturalized E. adeno- to the chemical conditions in differently aged leaves. phorum. However, a number of previous studies have Therefore, short colonization time trends may be the shown that endophytes of E. adenophorum tend to be major reason for the low diversity of fungi in young generalists. For example, Alternaria species are mainly leaves of E. adenophorum. Based on the changes in saprophytic fungi (Thomma 2003). Akimitsu et al. (2003) fungal diversity with increasing leaf age (Fig. 3), we consistently recovered A. alternata from healthy rough conclude that endophytic fungi in E. adenophorum can lemon tissue of asymptomatic leaves and found the accumulate on leaves shortly after emergence (about isolated strains to be unrelated to isolates causing disease. 7 days) and that infection densities increase steadily, A study also indicated that the most frequent genera of reaching a constant level after nearly 1 month (i.e., the endophytes were Alternaria from invasive spotted knap- fourth pair of leaves). weed (Centaurea stoebe, Asteraceae) in its native and Within a leaf, the distribution of diverse fungal species invaded ranges (Shipunov et al. 2008). Moreover, Clado- generally resembles a quilt-like patchwork, with different sporium is also dominant in fungal endophytes associated species usually abutting each other, producing an extremely with Citrus limon (Durán et al. 2005) and in rice (Shankar heterogeneous mix of various fungal species and genotypes Naik et al. 2009). Nevertheless, some endophytes were at very fine scales within the leaf matrix (Herre et al. 2007). found to be phylogenetically distinct from identified and In our study, the use of ONFIT allowed us to observe cultured fungus in GenBank (e.g., type X). However, we directly the heterogeneous distribution of species where a cannot conclude whether these are specific to E. adeno- few of the colonies were overlapping (data not shown). We phorum. Regardless of their specificity, it is evident that also found that each species showed a preference for some HTEs facilitate the host’s resistance response to differently aged leaves, with type X dominate in young pathogenic attacks (Arnold et al. 2003). The functions of leaves, type D flourishing in old leaves, types H and type J such endophytes in providing resistance to pathogens and coexisting in the third pair of leaves, type A dominate in the herbivores may be useful for exploring the invasive fourth pair of leaves, and type B dominate in the fourth and mechanisms of E. adenophorum. fifth pair of leaves (Fig. 3a). Subsequent experiments indicated that the heterogeneous Acknowledgments We thank the anonymous reviewer for detailed and constructive suggestions for our paper. This project was distribution and changes in diversity with increasing leaf financially supported by National Basic Research Program of China age are balanced by leaf chemistry and interactions between (973 Program) (2007CB411600), National Science Foundation of coexisting species. For example, types A and X were China (No. 30960077) and the Department of Science and Technology significantly inhibited by leaf chemistry (Fig. 4); however, of Yunnan Province, China (No.2007PY01-24). most interactions between coexisting species were positive, either through the mediation of volatile or non-volatile metabolites. All species promoted the growth of type X References through non-volatile metabolites, with the exception of type H (Table 1, 2). 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Journal

Annals of Microbiology – Springer Journals

Published: Jan 10, 2011

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