World Journal of Microbiology & Biotechnology 20: 303309, 2004. 2004 Kluwer Academic Publishers. Printed in the Netherlands.

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Study on the communities of endophytic fungi and endophytic actinomycetes from rice and their antipathogenic activities in vitro
X.L. Tian1, L.X. Cao1, H.M. Tan1, Q.G. Zeng1,2, Y.Y. Jia1, W.Q. Han1 and S.N. Zhou1,* 1 Department of Biochemistry, State Key Laboratory for Biocontrol, School of Life Sciences, Zhongshan University, Guangzhou 510275, P.R. China 2 College of Life Sciences, Jiangxi Normal University, Nanchang 330027, P.R. China *Author for correspondence: Tel.: 86-20-8411-0238, Fax: 86-20-8403-6215, E-mail: lsszsl@zsu.edu.cn
Received 13 August 2003; accepted 25 November 2003

Keywords: Endophytic actinomycetes, endophytic fungi, rice, rice blast disease, Sheath Blight of Rice

Summary The populations of endophytic fungi and actinomycetes from four rice cultivars in the Panyu district (Site 1) and Wushan district (Site 2) in Guangdong province, South China, were studied. The preponderant endophytic fungi and actinomycetes isolated belonged to Fusarium and Streptomyces respectively. The incidence of Streptomycetes griseofuscus ranged from 36.1 to 69% out of all the dierent rice cultivars from the two sites. It is the commonest population of endophytic actinomycetes, and constituted the greatest part of all the antagonistic communities. The distributions of endophytic fungi and actinomycetes in roots and leaves were dierent, endophytic fungi from leaves were diverse, some were organ-specic. More diverse endophytic actinomycetes were isolated from roots than from leaves. The endophytic fungi isolated from rice in Site 2 were more diverse than that in Site 1. The diversity of the endophytic actinomycetes, however, was less than that in Site 1. Acid soil in Site 2 is ideal for the growth and colonization of fungi while the alkaline soil in Site 1 is better for the growth and colonization of actinomycetes. The results suggested that dierences in the chemical composition of soil could inuence the endophytic microbial communities of rice plants. The endophytic fungi and actinomycetes isolated from poor-growing seedlings and susceptible rice cultivars were more abundant than that the disease-resistant counterparts. In the dual culture and activity detection of the metabolites, 41.2% of all the isolated endophytic fungi showed antagonism to rice pathogens. Fifty percent of all the isolated endophytic actinomycetes were antagonistic to those pathogens. The percentage of Streptomyces griseofuscus and hygroscopicus reached 55.4 and 21.4% of all the active actinomycetes.

Introduction Endophytes living asymptomatically within plant tissues have been found in many plant species. Endophytes can have many eects on their host such as enhancement of stress-, insect- and disease-resistance (Elzik 1985; Bush et al. 1997; Clay & Holah 1999; Shimizu 2000), productivity improvement (Quaroni et al. 1997; Shimizu et al. 2001), and herbicide activities (Peters et al. 1998) when in association with their hosts. The colonization and propagation of endophytes and their secondary metabolites inside the plants may be critical for these eects. These facts indicate that endophytes can be potential biological control agents and will play an important role in ecological agriculture. Endophytes also constitute a valuable source of secondary metabolites for the discovery of new potential therapeutic drugs (Miller 1995). Endophytic fungi were rst found in pasture plants (Breen 1994). Redman et al. (1999) proved that inoculating watermelon and cucumber seedlings with non-pathogenic endophytic Colletotri-

chum magna could rapidly induce systematic defence responses of plants, such as the production of peroxidase, phenylalanine ammonia-lyase, lignin, and salicylic acid. Formally, endophytic actinomycetes have been extensively studied only for the nitrogen-xing Frankia species from non-leguminous plants. Sardi et al. (1992) isolated endophytic actinomycetes from the roots of 28 plant species, most of them belong to Streptomyces. Endophytic actinomycetes were found to produce at least three kinds of antagonistic substances in plant tissues, including antibiotics, enzymes and siderophores (Trejo-Estradat et al. 1998). Recently Coombs research group in Australia have tested around 60 actinomycete species isolated from the root of wheat and identied some species which can kill the fungus responsible for take-all disease. These species can reduce the impact of take-all on wheat by up to 70% (Coombs 2002). To date there is no report on endophytic actinomycetes biocontrol on other agricultural crop diseases. Rice, one of the primary gramineous crops, constitutes the main nutrient resource for 40% of the worlds

304 population including most developing countries. But the yield is at serious risk when threatened with such diseases as Rice blast disease, Sheath Blight of Rice, Bacterial Leaf Blight and Rice Bakanae disease. Abuse of chemical pesticides, which are the most common approach for control, can destroy the balance of ecosystems and the contamination by their toxic residues may cause harm to humans and domestic animals. In addition, chemicals can decrease the soil microora sharply and induce tolerance of the pathogens. To reduce these negative eects, we hope to nd an ecofriendly and easily obtainable alternative to substitute for chemical pesticides. As an environmentally benign agent, endophytes are the ideal candidates. Study of rice endophytes has been mostly focused on endophytic diazotrophic bacteria, previous work has demonstrated their diversity, nitrogen-xing potential, the infecting pathways and their distribution and colonization in plants (Barraquio et al. 1997; Suman et al. 2001; Verma et al. 2001). Several species of fungal endophytes have been found on rice leaves (Fisher & Petrini 1992; Gonzales et al. 2000). So far, no studies have been undertaken to assess the incidence of endophytic actinomycetes of this important species. The present work was carried out to isolate endophytic fungi and actinomycetes from roots and leaves of rice in south China, and some strains antagonistic to rice pathogens have been screened. Tested pathogens

X.L. Tian et al.

Magnaporthe grisea and Rhizoctonia solani were isolated from infected rice, Xanthomonas oryzae pv. oryzae and Fusarium moniliforme were provided respectively by Guangdong Academy of Agricultural Sciences and Guangdong Institute of Microbiology.

Isolation of endophytes Isolation of fungi Surface sterilization was applied as described by Schulz et al. (1993), The surface-sterilized samples were cut into 1 cm fragments. Ten fragments from dierent parts of leaves and roots respectively per rice sample were placed on 1.2% PDA (Merck, 10130). Fifty microgram streptomycin (AMRESCO)/ml was added to repress bacteria. These plates were incubated at 25 C for 36 days. Hyphal tips of the developing fungal colonies were transferred onto PDA. After purifying the isolates several times, the nal pure cultures were transferred to PDA slant tubes.

Material and methods Sample collection Sample collection sites: Site 1 Panyu district, Guangdong province. Site 2 Wushan district, Guangdong province. Varieties of rice plant used in this study: Qilisimiao (Trade name), designated as Q-1. Huajingxian (Trade name), designated as H-1. Huaza35 (Trade name), designated as H-35. Jinfengzhan (Trade name), designated as J-1. Q-1, H-1, H-35, J-1 in Site 2 were collected from the four plots of one experimental eld, and Q-1 in Site 1 were collected from a eld in Guangdong province. Q-1 and H-1 are widely cultivated in the Guangdong province of China. Ten rice plants of each rice cultivar without signs of any disease were collected in the heading stage, the samples were placed in an ice-box and processed within 4 h of collection. Media S medium (Ruan et al. 1990), Gause No. 1 medium (Yan 1992), rice-medium (rice cut into pieces and boiled for 30 min, then ltered to remove insoluble materials, 18.0 g agar was added per liter), potato-dextrose agar (PDA).

Isolation of endophytic actinomycetes Samples were surface-sterilized as described in the Frankia isolation by Ruan et al. (1990). The samples were washed in running water and then immersed in 70% ethanol solution (5 min), 0.87% NaClO solution (15 min) and 10% NaHCO3 (15 min) in turns. Finally, they were rinsed three times in autoclaved distilled water. The surface-sterilized samples were cut into 1 cm fragments. Ten fragments from dierent parts of leaves and roots respectively per rice sample were placed on 1.2% S medium supplemented with 25 ppm K2Cr2O4 and 15 ppm nalidixic acid to repress bacteria and fungi. These were incubated at 27 C for several days. Every aerial mycelium tip of the developing actinomycete colonies was transferred onto S medium and incubated for 68 days, then the culture was puried by lining on the plate. Eventually puried cultures were transferred to G No. 1 medium slant tubes. Checking surface-sterilization The method was according as Song et al. (1999), a viability test was adopted to test the eectiveness of surface-sterilization (Schulz et al. 1993). Identication of isolates Identication of fungi Isolates from slants were transferred to PDA medium (about 100 ll) on a sterile microscope slide, which was then covered with a sterile cover-glass, incubated at 26 C and periodically the fungal fruiting structures were observed by microscope. Classication of fungi was according to Wei (1979).

Endophytic fungi and actinomycetes from rice Identication of actinomycetes This was according to Yan (1992), Ruan et al. (1990) and Zheng et al. (2000). Activity detection of endophytes and their metabolites Dual culture Pathogens and endophytes were inoculated on the centre and border of the rice-medium plate respectively, after incubation at 25 C, the antagonism was expressed by measuring the magnitude of interactions that could appear. Detection of metabolite activities (1) Extraction of fermented broth with organic solvents: all the endophytic fungi were inoculated in Potatodextrose liquid medium and incubated with 140 rev/min shaking at 25 C for 1 week. Endophytic actinomycetes were incubated in Gause No. 1 liquid medium with 180 rev/min shaking at 27 C for 10 days. All the fermented broth was freeze-dried and then supplemented with 5 ml methanol. (2) Activity detection: the pathogens were inoculated in the centre of the ricemedium plate, a sterilized lter paper was dipped into the extract and then placed around the pathogens on the plate. The colonies of the pathogens were observed after incubation for 35 days. Main types of endophytic fungi

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Results Eectiveness of the surface-sterilization The ow-out after washing the surface-sterilized samples (10 leaves and 10 roots) with sterilized water was plated on PDA plate and S plate, after incubated at 25 C for 2 weeks, no colony was found on both plates. Results of viability tests showed that 20 viable fungal and actinomycetic cultures (10 from leaves and 10 from roots) were killed by surface sterilization. It showed that epiphytes had been eradicated and the isolates were endophytes, present in plant tissues.

Altogether 72 endophytic fungi, mainly belonging to Fusarium, Pyricularia Sacc and yeasts were isolated from the cultivars in Site 1; 366 endophytic fungi were isolated from the cultivars in Site 2. Except for the dominating genera Fusarium, Penicillium and Aspergillus, genera Paecilomyces, Pyricularia Sacc. Helminthosporium and yeasts were also isolated (Table 1). Among dierent cultivars in Site 2, Q-1 and H-1 are relatively disease-resistant while H-35 is very susceptible to Magnaporthe grisea, Rhizoctonia solani and Xanthomonas oryzae pv. oryzae. J-1, a native cultivar in Guangxi province of China, is not adopted in Guangdong and grows poorly. More dierent endophytic fungi were isolated from H-35 and J-1 (more than ve and six genera respectively) than from resistance cultivars Q-1 and H-1 (more than 3 genera). The endophyte diversity dierence between Q-1 cultivars in Sites 1 and 2 implied that the distribution of endophytic fungi has an intimate connection with their host habitat. Fusarium is the commonest population from dierent cultivars in Sites 1 and 2, signicant dierences (99% condence limit) was found between Fusarium sp. and other endophytic fungi. The eect of endophytic Fusarium sp. during the developmental stage of rice deserved attention and further study. Endophytic fungi from rice plants in Site 2 were investigated further due to their species abundance. Of all the 366 isolated endophytic fungi species, 193 strains were from leaves, 173 strains from roots. Fusarium is the most abundant population, 40.9% in leaves, 56.9% in roots (P < 0.01) (Table 2). In leaves, the isolated frequencies of Pyricularia sp., Helminthosporium sp. and Paecilomyces sp. were 13.4, 6.0 and 6.1% respectively, but they do not exist in roots. It implied that these fungi are organ-specic. Pyricularia Sacc., Helminthosporium sp. and some strains of Fusarium sp. are pathogens of Rice blast disease, Rice brown spot and Rice Bakanae disease respectively. No symptom was found in the collected samples, implying that these fungi may be latent and may induce disease symptom when immunity declines due to plant tissues aging or outer stimulation

Table 1. Percentage of endophytic fungi population from rice plants in the Sites 1 and 2 (%)a. Endophytic fungi Sample collection sites and rice cultivars Site 1 Q-1 Fusarium sp. Penicillium sp. Aspergillus sp. Paecilomyces sp. Pyricularia Sacc. Helminthosporium sp. Yeast not identied Sterile mycelium
a

Site 2 Q-1 50.8 19.5 27.7 0 0 0 0 2.1 H-1 47.7 33.0 17.0 0 0 0 0 2.3 H-35 49.8 7.0 23.0 2.5 12.5 0 0 5.2 J-1 46.2 0 10.8 8.3 11.5 10.5 6.5 6.2

63.7 0 0 0 18.7 0 17.7 0

Ten plants of one cultivar, for each plant, 10 leave fragments and 10 root fragments were analysed.

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Table 2. Proportion of endophytic fungi isolated from leaves and roots of rice plantsa. Communities of endophytic fungi Organs of rice plant Leaves (%) Fusarium sp. Penicillium sp. Pyricularia Sacc. Aspergillus sp. Helminthosporium sp. Paecilomyces sp. Yeasts not identied Sterile mycelium 40.9 14.0 13.4 11.4 6.0 6.1 3.7 4.5 Roots (%) 56.9 11.5 0 27.6 0 0 0 4.0

X.L. Tian et al. were further identied as hygroscopicus, griseofuscus, aureus, globisporus, griseorubroviolaceus, albosporus, roseosporus, cinereus, glaucus. Compared with the endophytic actinomycetes from Q-1 in Site 1, only Streptomyces griseofuscus, S. hygroscopicus and S. griseorubroviolaceus were isolated from Q-1 in Site 2, it can be deduced that dierent growth environments may be responsible for the distribution of endophytes. The incidence of griseofuscus of Streptomyces ranges from 36.1 to 69% in all the dierent rice cultivars at the two sites, which are the preponderant endophytic actinomycete communities in Site 2 (P < 0.01). The occurrence of hygroscopicus of Streptomyces is also high but none of them was isolated from J-1 in Site 2. Further analysis on the distribution of endophytic actinomycetes in roots and leaves of the samples in Site 1 was conducted due to its higher diversity. Dierences in the distribution of endophytic actinomycetes in roots and in leaves were observed: Streptomyces aureus, S. globisporus, S. albosporus, S. roseosporus and S. glaucus were only present in roots and not in leaves. Five endophytic actinomycetes present in leaves were also found in the roots. Further analysis showed that 23 and 10 actinomycetes were isolated from roots and leaves respectively (Table 4). The relatively abundant diversity in roots may be caused by direct contact with the soil. Most endophytic actinomycetes from the samples in Site 1 were griseofuscus and hygroscopicus of Streptomyces. The percentage of Streptomyces griseofuscus (45.7%) was higher in the leaves than that of Streptomyces griseofuscus in roots (P < 0.05). The percentage of other communities except griseofuscus and hygroscopicus of Streptomyces were evidently lower in roots and leaves. Dual culture and detection of metabolite activities All the species of isolates and their metabolites were tested for anti-pathogen activity. Many strains and metabolites of some strains were found to have

a Ten plants, for each plant, 10 leave fragments and 10 root fragments were analysed.

(Schulz et al. 1993). The occurrences of Fusarium sp., Aspergillus sp. and Penicillium sp. common in the soil were 56.9, 27.6 and 11.5% in rice roots respectively. These fungi inside plants may come by the transmission from soil to plants. Main endophytic actinomycetes population Altogether 274 endophytic actinomycetes strains were isolated from four rice cultivars in Site 2, most of them belonged to Streptomyces (species griseofuscus, hygroscopicus, griseorubroviolaceus, cinereus, albosporus, aureus), only a few fell into Streptoverticillium. As with endophytic fungi, more endophytic actinomycetes were isolated from susceptible rice cultivars and poor-growing seedings than from resistant-cultivars: more than six actinomycetes populations were isolated from susceptible H-35 and poor-growing J-1 while only three populations were from resistant-cultivars, Q-1 and H-1 (Table 3). One hundred and ninety one endophytic actinomycetes strains were isolated from the Q-1 rice samples in Site 1 in all, 185 were identied as Streptomyces and 6 were Streptoverticillium. Those Streptomyces species

Table 3. Percentage (%) of endophytic actinomycetes genera isolated from rice plants in Site 1 and in Site 2a. Endophytic actinomycetes Sample collection sites and rice cultivars Site 1 Q-1 Streptomyces griseofuscus hygroscopicus griseorubroviolaceus cinereus albosporus aureus globisporus roseosporus glaucus Streptoverticillium sp.
a

Site 2 Q-1 H-1 H-35 J-1

36.1 33.5 6.8 3.1 1.6 6.8 6.3 1.6 1.0 3.1

63.4 26.8 9.8 0 0 0 0 0 0 0

54.4 33.0 0 12.6 0 0 0 0 0 0

48.2 8.7 8.7 9.6 17.1 0 0 0 0 7.7

69.0 0 4.6 4.9 3.3 13.0 0 0 0 4.9

Ten plants of one cultivar, for each plant, 10 leave fragments and 10 root fragments were analysed.

Endophytic fungi and actinomycetes from rice

Table 4. Quantities (Q) and percentage (%) of endophytic actinomycete from rice leaves and rootsa. Proportion of endophytic actinomycetes Roots Number of species Streptomyces griseofuscus hygroscopicus aureus globisporus griseorubroviolaceus albosporus roseosporus cinereus glaucus Streptoverticillium Total
a

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Leaves Q % Number of species Q %

5 5 3 2 1 1 1 1 1 3 23

32 35 13 12 5 3 3 2 2 3 110

29.1 31.8 11.8 10.9 4.5 2.7 2.7 1.8 1.8 2.7 100

4 3 0 0 1 0 0 1 0 1 10

37 29 0 0 8 0 0 4 0 3 81

45.7 35.8 0 0 9.9 0 0 4.9 0 3.7 100

Ten plants, for each plant, 10 leave fragments and 10 root fragments were analysed.

Figure 1. Dual culture between endophytes and rice pathogens. (A) Endophytic fungi Q-17 antagonistic to Magnaporthe grisea (Q-17 belonged to Fusarium sp.). (B) Endophytic actinomycete J-53, J-11 antagonistic to Rhizoctonia solani (J-53, J-11 belonged to Streptomyces griseofuscus). (C) Endophytic actinomycete f55, f166 antagonistic to Fusarium moniliforme (f55, f166 belonged to Streptomyces globisporus). (D) Metabolites of endophytic actinomycete f50, f27, f16 antagonistic to Magnaporthe grisea (f50, f27 belonged to Streptomyces hygroscopicus and f16 belonged to Streptomyces griseofuscus). Pathogens (Magnaporthe grisea, Rhizoctonia solani, and Fusarium moniliform) and endophytes (Q-17, J-53, J-11, f166, f55, f50, f27, f16) were inoculated on the centre and border of the rice-medium plate respectively, after incubation at 26 C, the antagonism was expressed by the magnitude of interactions that could appear. Detection of metabolite activities was shown in (D). All the fermented broth of isolated endophytes was freeze-dried and extracted with organic solvents; the pathogens were inoculated in the centre of the rice-medium plate, a sterilized lter paper was dipped into the extract (f50, f27, f16) and then placed around the pathogens on the rice-medium plate. after incubation at 26 C, metabolite activities was observed.

antagonism against Magnaporthe grisea, Rhizoctonia solani, Xanthomonas oryzae pv. oryzae. and Fusarium moniliforme (Figure 1). About 41.2% of all the isolated endophytic fungi showed antagonism against at least

one rice pathogen. These antagonistic fungi belong to Fusarium sp. (58.8%), Penicillium sp. (20.0%) and Aspergillus sp. (18.5%), signicant dierences (99% condence limit) was found between Fusarium sp. and

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Table 5. Population of antagonistic endophytic fungi and antinomyces against dierent rice pathogens. Rice pathogens Population of antagonistic endophytic fungi (total 100%) Fusarium Penicillium Aspergillus Sterile mycelium Magnaporthe grisea Rhizoctonia solani Xanthomonas oryzae pv. Oryzae Fusarium moniliforme Above four pathogens 54.1 60.0 100 59.3 58.8 22.3 29.4 0 35.0 20.0 23.6 7.1 0 5.7 18.5 0 3.5 0 0 2.7 Population of antagonistic endophytic actinomycetes (total 100%)

X.L. Tian et al.

Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces griseofuscus hygroscopicus globisporus aureus albosporus 54.5 53.1 66.7 50.0 55.4 21.8 24.5 33.3 25.0 21.4 7.3 8.2 0 10.0 7.1 9.1 8.2 0 7.5 8.9 7.3 6.1 0 7.5 7.1

other antagonistic endophytic fungi. Fifty percent of all the isolated endophytic actinomycetes showed antagonism against at least one rice pathogen. The percentage of Streptomyces griseofuscus and Streptomyces hygroscopicus reached 55.4 and 21.4% of all the active actinomycetes, signicant dierences (99% condence limit) was found between S. griseofuscus and other antagonistic endophytic actinomycetes (Table 5). Fusarium sp. and S. griseofuscus constituted the greatest part of all the antagonistic communities. Fifty ve percent of all the isolated S. griseofuscus have antagonism against rice pathogens. Sixty percent of all the isolated S. hygroscopicus and 50% of all the isolated S. globisporus showed strong antagonism. Those endophytic actinomycetes may play an important role in protecting the plant host against pathogenic microorganisms. Some metabolites of endophytic actinomycetes can strongly inhibit rice pathogens and may be a resource for new fungicides.

Discussion A pilot study on the population and distribution of endophytes from dierent cultivars at two sites in Guangdong province was conducted. Endophytic fungal species of rice were more abundant in Site 2 (more than 6 genera) than in Site 1 (3 genera). More actinomycete populations were isolated from rice in Site 1 (10 populations) than in Site 2 (6 populations). Dierent soil environments were found to be responsible for this dierence: acid soil in Site 2 was ideal for fungi while the alkaline soil in Site 1 was better for actinomycetes. Fusarium was the most frequently observed endophytic fungus in rice. The occurrence of the same endophytic fungus was dierent in the root and leaves of rice. Some endophytic fungi were organ-specic, which was also demonstrated in the research of Lepanthes (Bayman et al. 1997). Most of the endophytic actinomycetes were classied as Streptomyces, which was consistent with the result of endophytic actinomycetes from other plants (Sardi et al. 1992; Cao et al. 2003). Streptomyces griseofuscus was the commonest population of endophytic actinomycetes and constituted the greatest part of all the antagonistic community. Nota-

bly, for the rice in Site 1, the occurrence of Streptomyces hygroscopicus was very high (33.5%), close to that of Streptomyces griseofuscus (36.1%). This may be the result of dierent soil types. Endophytic actinomycetes from the roots were more diverse than from the leaves. From the four cultivars in Site 2, more endophyte species were found in poor-growing seedlings and susceptible rice cultivars than in disease-resistant and vigorous counterparts. Community analysis of endophytes in banana and potato have also demonstrated increased endophyte diversity in infected ones (Reiter et al. 2002; Cao 2003). In conclusion, soil types, rice cultivars, plant physiological status may account for the distribution of endophytes, further reasons remain to be explored. Most of active endophytic fungi showed evident antagonism in the dual culture test, whereas their metabolites showed no or little activity. This implies that no spreadable antibiotics were produced when they interacted with the pathogens, and that other antimicrobial mechanisms may be involved. Most of the active endophytic actinomycetes in the isolates belong to griseofuscus and hygroscopicus of Streptomyces. Many strains of hygroscopicus have the character of higheectiveness, extensive spectrum, permanence and steadiness when they act against pathogens, which are promising resources for biologically active compounds. These active endophytes inside the plants have intimate correlation with the development and physiological activity of rice. The distribution and biological activity of these endophytes deserve to be explored to make full use of their habitation inside plants. Acknowledgements This work was supported by grant from the Natural Science Foundation of Guangdong (011124), China and the project of Research and Development (211), China. References
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