Diversity and plant growth-promoting ability of endophytic fungi from the five flower plant species collected from Yunnan, Southwest China

Abstract

The diversity and plant growth-promoting ability of endophytic fungi associated with the five flower plant species growing in Yunnan, Southwest China, were investigated. A total of 357 culturable endophytic fungi were isolated from 1000 segments of healthy leaves and stems of the five plant species. Based on the morphological characteristics and the rDNA internal transcribed spacer (ITS) analysis, the isolates were identified to 24 taxa, of which Alternaria, Phomopsis, Cladosporium, and Colletotrichum were the dominant genera. The Sorenson's coefficient similarity indices of the endophytic fungi from the five flower plant species ranged from 0.36 to 0.80. It was found that the similarity index between two cultivated flowers (0.8) or the similarity index between two wild flowers (0.71–0.76) was higher than the similarity index between one cultivated flower and one wild flower (0.36–0.48). The Shannon indices (H) of the endophytic fungi from the five plant species ranged from 1.73 to 2.45, and the diversity indices of the wild flowers were higher than those of the cultivated flowers. The plant growth-promoting tests indicated that some isolates could improve the host plants' growth more efficiently when compared with the control (p < 0.05, least significant difference test).

Keywords:

• endophyte
• fungal diversity
• flower plant
• tobacco
• growth-promoting

Introduction

The fungi causing asymptomatic infections in living plant tissues have been called endophytic fungi (Hyde & Soytong 2008). They have been widely studied in various geographic and climatic zones and were found to be ubiquitous within plant tissues and rich in species diversity (Ghimire et al. 2011; Rivera-Orduña et al. 2011; Li et al. 2012b; Tanwar & Aggarwal 2013). It has been demonstrated that endophytic fungi play important roles in providing nutrients to hosts, adapting hosts to their environments, defending hosts from biotic and abiotic stresses, and promoting plants community biodiversity (Kharwar et al. 2008; Berg 2009; Gond et al. 2010; Pandey et al. 2011; Li et al. 2012a).

For its low latitude, high altitude, complex landform, and diversiform climate type, Yunnan (97°31′–106°11′ N, 21°8′–29°15 E′) is a very suitable place for the flower plant growth and is known as the garden of flowers in China (Qin & Yang 2008; Liu & Shen 2010). It is also the most important cut-flower planting base in China and about 50% of cut-flowers were supplied by Yunnan each year. To improve the production of flowers, various fertilizers were used in floriculture in the past decades, which created a series of environmental problems (Casey et al. 2007; Gunnell et al. 2007; Leach & Mumford 2008). In comparison with the chemical/synthesized fertilizer, endophytic fungi have several characteristics that make them an ideal candidate for biofertilizers in floriculture in the greenhouse (Murphy et al. 1998; Berg 2009; Campanelli et al. 2013).

Although there have been a few reports on the diversity and function of endophytic fungi of the wild flower growing in Yunnan (Miao et al. 2011; Chen et al. 2012; Li et al. 2012b; Tan et al. 2012), the diversity and its ecological roles of endophytic fungi of the cultivated flower as well as the difference of fungal diversity between the wild flower and the cultivated flower have never been reported until now. In the present study, the diversity of endophytic fungi of three wild flowers and two cultivated flowers as well as their plant growth-promoting ability were investigated. The results would not only provide a new insight into the fungal diversity of the cut-flower planted in the greenhouse but also contribute to understand the possible roles of endophytic fungi associated with the flower plant.

Materials and methods

Study site and sample collection

Rosa rugosa, Camellia japonica, and Delonix regia growing in wild, while Dianthus caryophyllus and Rosa hybrid planted in the greenhouse were collected from Yunnan, Southwest China in May 2012. Among them, R. rugosa, R. hybrid, and D. caryophyllus were collected from Dounan, Kunming (102°78′ N, 24°89′ E), C. japonica was collected from Jingdian, Kunming (102°76′ N, 25°08′ E) and D. regia was collected from Xinping county (101°99′ N, 24°07′ E). For each plant species, 15 healthy individuals at least 10 m apart from each other were chosen, and three healthy and separate branches from each plant were collected at random. All samples were brought to laboratory in sterile polythene bags and processed within 24 h.

Fungal isolation, culture and identification

For the isolation of endophytic fungi, 15 healthy stems and 15 healthy leaves were selected from each plant species at random, washed in running tap water, and processed as follows: the samples were cut into segments (about 5 × 5 mm) and surface sterilized by sequentially dipping into 0.5% sodium hypochlorite (2 min) and 70% ethanol (2 min; Li et al. 2012b). Then, 100 leaf segments and 100 stem segments from each plant species were placed in a Petri dish containing potato dextrose agar (PDA) medium amended with 0.5 g l⁻¹ streptomycin sulfate, incubated at 25°C, and checked every other day for 45 days. The fungi growing out of the plant tissues were transferred to fresh PDA plates. The effectiveness of the surface sterilization was controlled by making imprints of disinfected segments on PDA plates (Schulz et al. 1998).

The fungal identification was based on the morphology of the colony, the mechanism of spore production, and the spore characteristics formed in PDA or autoclaved carnation leaves in water agar (Sutton 1980; Barnett & Hunter 1998; Ellis 1988). Sterile isolates were sorted into different groups on the basis of colony surface texture, hyphal pigmentation, margin shapes, and growth rates. Twenty representative isolates from 10 morphological taxa (2 for each taxon) were further identified based on their rDNA internal transcribed spacer (ITS) sequence analysis, noting that matches in GenBank did not necessarily give correct names (Ko Ko et al. 2011). All of the isolates were deposited in the Faculty of Life Sciences and Technology, Kunming University of Science and Technology.

Screening for plant growth-promoting endophytes

To obtain the sterile seedlings, the tobacco (Nicotiana tabacum) seeds were surface sterilized by dipping into 75% ethanol (2 min) and 10% sodium hypochlorite (2 min), then, washed with sterile water and planted onto the Murashige and Skoog medium (Murashige & Skoog 1962) supplemented with sucrose 15 g l⁻¹ and agar 8 g l⁻¹, and incubated at 25°C for germinating. The seedlings growing out were free of the fungal endophytes.

A total of 102 sterile tobacco seedlings of similar size were used to screen the plant growth-promoting endophytes. And 24 isolates of 6 dominant genera (Alternaria, Cladosporium, Colletotrichum, Phomopsis, Phoma and Ascomycetes, and 4 isolates from each taxon) were selected at random to test their plant growth-promoting ability as follows: to obtain the endophyte-inoculated seedlings, several fungal disks (diameter 0.5 cm) were cut from a fresh culture (4–7 days) of each isolate and then inoculated onto the roots of sterile seedlings. Control seedlings were inoculated using agar disks without the fungus. There were four seedlings were inoculated with each isolate. All seedlings were incubated at 25°C under a 14-h photoperiod.

Before transplanting into plastic pots, 24 seedlings inoculated with 24 different endophytes and 3 control seedlings were selected at random to test the success of inoculation. Positive infected seedling was verified by isolating the strain, which was used to inoculate the seedling before, from the surface-sterilized root and/or shoot of the tested seedling, whereas, the same fungus was not isolated from the control seedlings (Li et al. 2012a).

There were three replicates for each treatment. The height of the seedlings was measured and recorded every 10 days for 50 days. The isolates which could improve the growth of tobacco seedlings significantly were selected to conduct the following pot experiments.

Pot experiments

The isolates H25 (Phomopsis), B50 (Cladosporium), A38 (Alternaria), and A7 (Alternaria), which showed better plant growth-promoting ability in above experiments, were selected to carry out pot experiments.

A total of 40 sterile tobacco seedlings of similar size were selected. For each isolate, eight sterile tobacco seedlings were inoculated, and eight tobacco seedlings inoculated with agar disks without the fungus were served as the control check (CK). All treatments were incubated at 25°C under a 14-h photoperiod for 4 weeks. Then, eight seedlings inoculated with endophytes (two seedlings for each strain), and two control seedlings were selected at random to test the success of inoculation as the method described earlier. Finally, all of the endophyte-inoculated seedlings and control seedlings were transplanted into the plastic pots (26 cm diameter × 21 cm height, three seedlings in each pot) containing 6 kg river sand and perlite (v/v, 2:1). Before putting into the plastic pots, the river sand was passed through a 0.5 mm sieve and mixed with perlite thoroughly and autoclaved at 121°C for 2 h for three times. There were six replicates for each strain. All experimental seedlings were cultured in a sunlit greenhouse with natural light, a 16/8-h day/night cycle at 25/18°C and 60–80% relative humidity. Seedlings were regularly watered with deionized–distilled water and each pot received 300 ml sterile Hoagland's nutrient weekly.

Plant harvest and chemical component test

After 2 months' growth, the plants were harvested and rinsed with deionized water and blotted up water with sterile filter paper. Then, the leaf area was measured and recorded (six replicates for each treatment), and 300 mg leaves were used to conduct the chlorophyll content analysis according to the method of Wellburn and Lichtenthaler (1984). Finally, the remnant seedlings were oven-dried at 80°C to measure dry weight, and then, the soluble sugar was performed as the method of Pirzad et al. (2010).

Data analysis

The colonization rate (CR) was calculated as the total number of plant tissue fragments infected by one or more fungi divided by the total number fragments incubated (Kumar & Hyde 2004). The relative frequency (RF) was calculated as the number of isolates of one species divided by the total number of isolates (Su et al. 2010; Yuana et al. 2011).

The endophytic fungal diversity was evaluated using the Shannon index, which has two main components, evenness and the number of species. The Shannon index (H) was calculated according to the following formula: , where k is the total species number of one plot and P_i is the relative abundance of endophytic fungal species in one plot (Spellerberg & Fedor 2003). To evaluate the degree of community similarity of the endophytic fungi between the two treatments, Sorenson's coefficient similarity index (Cs) was employed and calculated according to the following formula: Cs = 2j/(a + b), where j is the number of endophytic fungal species coexisting in two treatments, a is the total number of endophytic fungal species in one treatment and b is the total number of endophytic fungal species in another treatment (Su et al. 2010). The analysis of variance was used for data analysis of factorial experiments. The differences of plant biomass between endophyte-inoculated and uninocolated plants were determined by the least significant difference (LSD) test. And the rejection level was set at p < 0.05. The chi-squared test was used to compare the difference in the CR of the endophytes between the stem and leaf. All data were analyzed by SPSS 11.5.

Results

Composition of endophytic fungi

A total of 357 endophytic fungi were isolated from 1000 tissue segments of the five flower plant species (243 from the stems and 114 from the leaves). The CRs of the endophytic fungi from the five flower plant species ranged from 9.5% to 79.5%, and the CRs of the wild flowers (32.0–79.5%) were significantly higher than those of the cultivated flowers (9.5–13.5%; p < 0.05, LSD test; Table 1). The highest CR appeared in C. japonica (79.5%), whereas the lowest was found in R. hybrid (9.5%). The mean CR in the stems (48.2%) was significantly higher than that in the leaves (22.0%; p < 0.001, chi-squared test; Table 1).

Table 1. Number, CR, and Shannon index (H) of the endophytic fungi (EF) from the 5 flower plant species.

	No. of segments plated (No. of segments colonized by EF)			No. of EF isolated			CR (%)			H
Host plants	Stem	Leaf	Total	Stem	Leaf	Total	Stem	Leaf	Total	Total
C. japonica	100 (85)	100 (74)	200 (159)	85	78	163	85.0^a	74.0^a	79.5	2.45
R. rugosa	100 (68)	100 (14)	200 (82)	70	14	84	68.0^a	14.0^b	41.0	2.36
D. regia	100 (63)	100 (1)	200 (68)	63	1	64	63.0^a	1.0^b	32.0	1.95
D. caryophyllus	100 (15)	100 (12)	200 (27)	15	12	27	15.0^a	12.0^a	13.5	1.77
R. hybrid	100 (10)	100 (9)	200 (19)	10	9	19	10.0^a	9.0^a	9.5	1.73
Total	500 (246)	500 (110)	1000 (356)	243	114	357	48.2^a	22.0^b	35.1

Note: Values with different letters in the same row indicate significant difference at p < 0.05 level.

The endophytic fungi from the five flower plant species were identified to 24 taxa, of which Alternaria, Phomopsis, Cladosporium, and Colletotrichum were the dominant genera, and the RFs of them were 21.57, 17.37, 5.04, and 5.04%, respectively (Table 2). In contrast, some taxa were rare, and only a few isolates were obtained, such as Meria, Geotrichum, and Melanconium (Table 2). The numbers of taxa isolated from C. japonica, R. rugosa, D. Regia, D. caryophyllus, and R. hybrid were 20, 17, 14, 8, and 7, respectively. Overall, the wild flower harbored more fungal taxa than the cultivated flower, and 15 of the taxa only existed in the wild flower, such as Gloeosporium, Phomopsis, and Colletotrichum. Only two of the taxa coexisted in all five of the plant species, they were Alternaria and Phoma (Table 2).

Table 2. Numbers, taxa, and RFs of the endophytic fungi from the 5 flower plant species.

	No. of strains isolated from the 5 flower plant species (RF%)
Taxa	C. japonica	R. rugosa	D. regia	D.__caryophyllus	R. hybrid	Total (RF%)
Alternaria sp.^a	40 (24.54)	25 (29.76)	1 (1.56)	8 (29.63)	3 (15.79)	77 (21.57)
Aspergillus sp.1	–	2 (2.38)	–	1 (3.70)	2 (10.53)	5 (1.40)
Aspergillus sp.2	1 (0.61)	1 (1.19)	–	–	–	2 (0.56)
Cladosporium sp.^a	4 (2.45)	6 (7.14)	–	5 (18.52)	3 (15.79)	18 (5.04)
Colletotrichum sp.^a	16 (9.82)	–	2 (3.13)	–	–	18 (5.04)
Geotrichum sp.	3 (1.84)	–	–	–	–	3 (0.84)
Gloeosporium sp.^a	4 (2.45)	4 (4.76)	8 (12.50)	–	–	16 (4.48)
Melanconium sp.	–	2 (2.38)	1 (1.56)	–	–	3 (0.84)
Meria sp.	–	–	–	1 (3.70)	1 (5.26)	2 (0.56)
Ovulariopsis sp.	2 (1.23)	–	4 (6.25)	–	–	6 (1.68)
Penicillium sp.	1 (0.61)	–	1 (1.56)	–	2 (10.53)	4 (1.12)
Pestalotia sp.	6 (3.68)	–	–	–	–	6 (1.68)
Phoma sp.^a	2 (1.23)	1 (1.19)	7 (10.94)	2 (7.41)	1 (5.26)	13 (3.64)
Phomopsis sp.1^a	–	5 (5.95)	27 (42.19)	–	–	32 (8.96)
Phomopsis sp.2^a	4 (2.45)	2 (2.38)	7 (10.94)	–	–	13 (3.64)
Phomopsis sp.3^a	7 (4.29)	9 (10.71)	1 (1.56)	–	–	17 (4.76)
Rhynchosporium sp.^a	1 (0.61)	10 (11.90)	–	–	–	11 (3.08)
Stemphylium sp.	3 (1.84)	–	–	1 (3.70)	–	4 (1.12)
Sterile mycelium I	6 (3.68)	3 (3.57)	2 (3.13)	2 (7.41)	–	13 (3.64)
Sterile mycelium II	4 (2.45)	5 (5.95)	1 (1.56)	7 (25.93)	7 (36.84)	24 (6.72)
Sterile mycelium III	6 (3.68)	1 (1.19)	1 (1.56)	–	–	8 (2.24)
Sterile mycelium IV	4 (2.45)	6 (7.14)	–	–	–	10 (2.80)
Sterile mycelium V	23 (14.11)	1 (1.19)	1 (1.56)	–	–	25 (7.00)
Ascomycetes (one species, unidentified)^a	26 (15.95)	1 (1.19)	–	–	–	27 (7.56)
Total	163 (100)	84 (100)	64 (100)	27 (100)	19 (100)	357 (100)
Number of taxa	20	17	14	8	7	24

Note: ‘–’ indicates the taxa were not found in plants. ‘a’ indicates that the taxa were further identified by molecular method but were not been accurate to species level.

The Sorenson's coefficient similarity indices (Cs) for the endophytic fungi from the five flower plant species ranged from 0.36 to 0.80 (Table 3). It was found that the similarity index between two cultivated flowers (0.8) or the similarity index between two wild flowers (0.71–0.76) was higher than the similarity index between one cultivated flower and one wild flower (0.36–0.48;Table 3). The Shannon indices (H) of the endophytic fungi from the five flower plant species ranged from 1.73 to 2.45. The diversity indices of the wild flowers were higher than those of the cultivated flowers (Tables 1 and 3).

Table 3. The Sorenson's coefficient similarities indices (Cs) of the fungal endophytes from the 5 flower plant species.

Plant	R. rugosa	R. hybrid	D. caryophyllus	C. japonica
R. hybrid	0.42
D. caryophyllus	0.48	0.80
C. japonica	0.76	0.37	0.43
D. regia	0.71	0.38	0.36	0.71

Plant growth-promoting ability of endophytic fungi

According to the method mentioned above, it was found that all of the endophyte-inoculated seedlings were positive infected, on the contrary, the control seedlings were negative infected.

As shown in Table 4, it was found that different isolate showed different growth-promoting ability. For example, the leaf area and the dry weight of the seedlings inoculated with A7 were significantly increased when compared with the control (p < 0.01, LSD test). However, the leaf area of the seedlings inoculated with B50 and A38 showed no significant changes. For the soluble sugar contents, all of the endophyte-inoculated seedlings were significantly increased in comparison with the control (p < 0.01, LSD test). Similarly, the chlorophyll contents of the seedlings inoculated with the endophytes were significantly increased (p < 0.01, LSD test), except for that inoculated with A7 (p < 0.05, LSD test).

Table 4. The biomass of seedlings inoculated or uninoculated with the endophytic fungi.

Treatments	Leaf area (cm^2)	Plant dry weight (g)	Chlorophyll (mg/g FM)	Soluble sugar (mg/g FM)
CK	35.51 ± 20.14	0.32 ± 0.09	104.62 ± 0.29	27.26 ± 1.59
H25	73.25^** ± 13.37	0.46^* ± 0.07	142.67^** ± 1.34	54.78^** ± 1.24
B50	38.19 ± 6.89	0.50^* ± 0.09	142.69^** ± 0.14	46.14^** ± 4.68
A38	40.39 ± 13.11	0.54^** ± 0.08	145.64^** ± 0.57	38.26^** ± 3.98
A7	70.55^** ± 22.85	0.52^** ± 0.05	121.73^* ± 0.57	52.32^** ± 7.54

Note: Values with ‘*’ or ‘**’ in the same column refer to significant difference at the 0.05 and 0.01 levels according to LSD test, respectively. FM means the fresh weight.

Discussion

CR

In the previous study, it was found that the CRs of the endophytic fungi in the wild flowers ranged from 54.0 to 100%. For instance, the CRs of 54.5–74.0% were found for three Rhododendron species from Baima Snow Mountain and 55.0–100.0% for C. japonica from the Oharano Forest Park of Kyoto City, Japan (Osono 2008; Li et al. 2012b). In the present study, the CRs of endophytic fungi in the three wild flowers ranged from 32.0 to 79.5%, which were very closed to those records. In contrast, it was found that the CRs of the endophytic fungi in the two cultivated flowers (9.5–13.5%) were significantly lower than those in the wild flowers (Table 1; p < 0.05, LSD test). This may be due to the various fertilizers, pesticides, and herbicides that were used in the cut-flower planting, which killed some endophytes or hindered their colonization. The other possible reason may be that the cultivated flower's community structure was relatively unitary, which gave less chance for endophytic fungal transmission among different plant species.

Composition of endophytic fungi

Alternaria and Phomopsis are very common and have been reported as the dominant fungal endophytes of various plant species in diverse environments (Huang et al. 2009; Chen et al. 2011a; Gond et al. 2012). In the present study, they were found to be the most dominant genera, too, and the RFs of them were 21.57 and 17.37%. However, it was found that Alternaria widely existed in both the wild flowers and the cultivated flowers. On the contrary, Phomopsis was found only existed in the wild flowers. The ecological function of Alternaria in the cultivated flowers need further study.

It has been demonstrated that some plants require symbiotic associations for the stress tolerance (Rodriguez & Redman 2008). In the present study, 15 of the 24 taxa only existed in the wild flowers, on the contrary, only 1 taxon (Meria) existed in the cultivated flowers. The possible reason may be that the cultivated flowers in the greenhouse were subjected to less selective pressures, therefore, their symbiont-dependent decreased. The other possible reason may be that various fertilizers, pesticides, herbicides, and so on, which were used in the cut-flower planting, killed most of the endophytes found in the wild flower.

The Sorenson's coefficient similarity index between two cultivated flowers or the similarity index between two wild flowers was higher than that between one cultivated flower and one wild flower. The result suggested that the endophytic fungal community was mainly influenced by the environmental factors rather than the host plant species. The similar result was reported by other researchers (Hoffman & Arnold 2008; Liu et al. 2010; Suryanarayanan et al. 2011).

Plant growth-promoting ability of endophytic fungi

It has been reported that different endophytes could enhance plant's growth through different mechanisms (El-Deeb et al. 2012; Hamayun et al. 2012). For instance, Chen et al. (2011b) found that the endophytic fungi Phomopsis liquidambari can produce enzyme to degrade the phenolic acid allelochemicals which have negative impacts on the growth of plants to alleviate the effects of the ecological stress; Waqas et al. (2012) reported that Phoma glomerata and Penicillium sp. could secrete gibberellins (GAs) and indoleacetic acid to significantly promote the growth of GAs-deficient rice, and Muthukumarasamy et al. (2002) reported that the nitrogen-fixing endophytes of sugarcane contribute abundant nitrogen to the plant and improve plant growth. In the present study, it was found that different strains showed different plant growth-promoting ability (Table 4). It was supposed that these isolates improve tobacco seedlings' growth through different ways. However, to understand the mechanisms of the endophytic fungi in plant growth promoting, it still needs more works.

Soluble sugars play an obviously central role in the plant structure and metabolism at the cellular and whole-organism levels. They are involved in the responses to a number of stresses, and they act as nutrient and metabolite signaling molecules that activate hormone transduction pathways (Couée et al. 2006). Chlorophyll is a key component of photosynthesis required for the absorption of sunlight and inhibition of chlorophyll biosynthesis can lead to some plants growth inhibition and cell death (Hörtensteiner & Kräutler 2011). In the present study, the contents of soluble sugar and chlorophyll of tobacco seedlings inoculated with the endophytes were significantly increased in comparison with the control (p < 0.01, LSD test; Table 4), which indicated that these endophytes are benefits to the hosts' growth. Therefore, these endophytes may have a potential use in floriculture to alleviate environmental stress and reduce agricultural cost in the future.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (31360128), the Natural Science Foundation of Yunnan province (KKS0201126013), the Science Foundation of Yunnan Educational Committee (KKJA2011 26033), and National Undergraduates' Innovative Entrepreneurial Training Program of China (2012). The authors thank to Professor Zhiwei Zhao for plant identification.