Isolation and Identification of the Causal Pathogens for Kiwifruit Bacterial Canker and the Isolation of the Antagonistic Endophytic Fungi From Kiwifruit in Sichuan, China

The kiwifruit bacterial canker has been recognized as the main kiwifruit pathogen in Sichuan and acquired strong resistance to chemicals during its long evolution under chemical evolutionary pressure. Base on biochemical testing, pathogenic testing and phylogenetic analyses, the results shown that Pseudomonas syringae pv. actinidiae is the causal agents of the “Hongyang” kiwifruit bacterial canker disease. Besides this bacterial pathogen, 14 endophytic fungi of kiwifruit leaves were also isolated and identified, including Penicillium sp. Colletotrichum sp. Phomopsis sp. Alternaria sp. and Nigrospora sp. 8 endophytic fungi were obtained from branches, such as Alternaria sp. Nigrospora sp. Phomopsis sp.and Sordaria sp. and one of the endophytic fungi Nigrospora sp. (No.J2) from branch showed good antagonistic activities to Pseudomonas syringae pv. actinidiae in vitro with the inhibition zone of 14 mm, therefore it has the potential of being used as a biological control agent against kiwifruit bacterial canker.


Introduction
China is one of the most important production areas for kiwifruit around the world and known as the country of origin of kiwifruit.Sichuan province, located in southwest of China, has a wealth of natural resources with about 700,000,000 m 2 of kiwifruit growing areas and an annual production of 84,000,000 kg where "HongYang" kiwifruit is especially characterized with red color, good taste, and rich nutrient, renowned as the "national variety protection resource" and has become the main kiwifruit specie in Sichuan with good economical benefits and cultural values.However, the kiwifruit bacteria canker constitute causing serious yield loss in this area.The main disease symptoms were brown spots surrounded by yellow haloes on leaves and reddish exudates on twigs and trunks (Figure 1) which were similar to those caused by the bacterium Pseudomonas syringae pv.actinidiae (Psa.) in previous reports (Serizawa et al., 1989).Several studies have reported the kiwifruit bacterial canker in other countries, but the comprehensive research on the pathogens of kiwifruit bacterial canker in "HongYang" is still seldom.
Endophytes are organisms able to live in plant tissues for a consistent part of their life cycle without inducing substantial damages to the host (Downing et al., 2000;Ryu et al., 2005).It is also known that endophytic fungi statement in abstract and introduction are an important source of bioactive compounds, some showing a real antifungal activity (Shimizu et al., 2000).As matter of facts, many have been already used as fungicide in a commercial way (Li et al., 2005).The outburst condition of kiwifruit bacterial canker in "HongYang" is especially serious in 2012, however, there is a particular kiwifruit plant still in healthy state while all of the kiwifruit plants surrounded were seriously affected.Therefore we have reason to suspect that maybe is the endophytic fungi in that particular plant that works.There are many researches on endophytic fungi in different plants, but no reports concern kiwifruit.
In this study, we isolated and identified the kiwifruit bacterial canker pathogen on "HongYang" from reddish and F3 (ACCTGGTGAAGTTGGTCAGAGC) / R4 (CGCACCCTTCAATCAGGATA).The sequences were then compared to the database available at the National Center for Biotechnology Information (NCBI) using their Blast Search Software on the NCBI website (http://www.ncbi.nlm.nih.gov/genbank).
The biochemical test was performed according to Lelliott & Stead (Lelliott & Stead, 1998), levan production, presence of oxidase, Gram staining, arginine dihydrolase, metabolism of glucose, presence of fluorescent pigment on King"s medium B, tobacco-hypersensitivity reaction.The effect of temperature on growth was measured according to the amount of the bacteria produced after being incubated for 24 h at different temperatures (Shanghai Yiheng light incubator MGC-250BP-2, Shanghai).The relationship between the growth and the time was monitored according to the amount of bacteria incubated at 28°C for different amount of times.Shape of the pathogen was observed by a scanning electronic microscope (Hitachi cold field emission scanning electronic microscope S-4800, Japan).
Three 2-year-old healthy plants were inoculated with about 1×10 7 CFU ml -1 of bacterium suspensions as described by Takikawa (Takikawa et al., 1989) as compared to water control.Only isolates bacterium reddish exudates from canes produced symptoms similar to those naturally observed on the buds and branches and identify the bacterium by molecular tests.The control plants were similarly incubated with sterile water.

Isolation and Identification of the Endophytic Fungi
In July 2012, branch and leaf samples of the particular healthy kiwifruit plant were taken and transported in plastic bags in a cooler from the field sites to the laboratory.The isolation was carried out as following: branches and leaves were washed under running tap water for 3 min and than air-dried.Samples were pieced into the size of about 0.5 cm using sterile surgical blades, soaked in 75% ethanol for 2 min, washed by sterile distilled water for 3 min, immersed in 0.1% HgCl 2 for 20 s, and then washed by sterile distilled water for 1 min.The treated pieces were cultured on the potato dextrose agar (PDA) medium (potato 200 g, glucose 17 g, agar 17 g, water 1000 ml) supplemented with streptomycin (100 μg/ml) sequentially.
Total DNA was extracted by CTAB method (Wu et al., 2009) and PCR was performed using an Eppendorf Master Cycler Gradient.The strains were detected by the primers ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC).The amplified DNA fragments were purified and the sequences were then compared to the database available at the NCBI using their Blast Search Software on the NCBI website (http://www.ncbi.nlm.nih.gov/genbank).

The Antibacterial Activity Test of the Endophytic Fungi
The antibacterial activity was determined using the oxford-cup-plate method (cited literatures).1×10 7 CFU suspension of PSA was dispersed on NA plates.Endophytic fungi were grown in 250 ml Erlenmeyer flasks containing 150 ml malt PDA liquid medium.The culture was grown with continuous shaking on a rotary shaker (180 rpm) at 26°C for 3 days.After incubation，endophytic fungi were harvested by a centrifugation at 5000 rpm for 10 min and 20 μl of endophytic fungi extracts were pipetted into a sterile oxford-cup (6 mm×10 min).These were placed on the surface of the plate and incubated at 30°C for 48 h.Sterile water was used as a control.Antibacterial activity was measured by the diameter of the inhibition zone.For the stains that showed good bacteriostasis, 7 generation was subcultured and the subculture was tested again to make sure the stability.

Results
Three strains from three sites were obtained.
For colony Dujiangyan, there was no obvious symptom on cane No.1 during the first 8 days.It produced the reddish exudates in Day 9 with water soaked lesions appeared around the wound.For colony Shifang it was until the 11 days that the cane appeared obvious symptom: reddish exudates produced on twigs.For colony Ya/an, it took 8 days to appear the symptom on cane No.3.Bacterium with morphological, biochemical and molecular characteristics identical to the original isolate were re-isolated from tissue showing symptoms.
According to the techniques reported by Lelliott and Stead (1988), these strains were positive for levan production, tobacco-hypersensitivity reaction and metabolism of glucose.Negative for presence of oxidase, potato soft rot, arginine dihydrolase, Gram staining and the presence of fluorescent pigment on King"s medium B.
The relationship between growth and temperature was analyzed.It was demonstrated that the most suitable temperature for bacterium growth is around 28°C (Figure 2).It grows slowly under 0°C to 4°C.

Figure
Figure 2. T