Etiology Of Mulberry Shrunken Popcorn Disease And Development Of A Molecular Detection System For The Pathogen

The mulberry popcorn disease is an important mulberry disease and is a main constraint of mulberry fruit production in China. The disease has been distinguished into three types according to the symptoms:swollen, shrunken and diminished fruits. Ciboria shiraiana, Ciboria carunculoides and Scleromitrula shiraiana were thought of as the pathogens inducing popcorn disease, but the relationship between the pathogens and disease types is still not clear and detailed information on morphology and biology of the pathogens was not easily traced and referable from literatures. The molecular method for detecting the pathogens and diagnosing the disease was still lacking. In the present study, the pathogen of shrunken popcorn disease was identified and the biology of the pathogen was investigated. A molecular system for detecting the pathogen was developed using SCAR-PCR.1. Isolation and identification of the pathogen causing shrunken-fruit popcorn disease. A total number of 247 samples were collected from different fruit mulberry orchards in Chongqing in 2012-2014 for isolation with 3 methods. It was failed to obtain the pathogen with the tissue isolation and spore germination method. The 25 diseased mulberry fruits (with sclerotia not yet formed) were wet-cultured till the mycelia grew out. The mycelia were then transplanted onto PDA plates and 5 isolates were obtained. These isolates were all identified as Scleromitrula shiraiana morphologically and molecularly and were diagnosed as the pathogen of shrunken popcorn disease via the Koch’s postulate procedures.2. Morphology of S. shiraiana. One or several stalked apothecia were formed from individual sclerotia. The stalks of apothecia were relatively long, tabular, grey-brown and were covered with villi. Heads of the apothecia were fusoid, with many lengthways gills. The asci were formed on the gills and they were sized 4-6μm×53-68μm. Eight ascospores were produced in each ascus; they were oval, colorless and transparent, measured 2.3-4.2 μm× 4.8-9.7 μm. On PDA plates, the colony was grey-white, with irregular edge. Sticky and thick conidial masses were produced at later stage of culturing, forming a tough layer on surface of the colony. The conidia were monocellular, colorless, oval or near-circular, measured 1.8-2.2 μm×4.3-4.9μm.3. Biological characteristics of S. shiraiana. Eight media were tested and the fungus grew best on mung bean dextrose agar (MBDA) and mulberry leaf dextrose agar (MLDA) plates. The growth on PDA plates was relatively slow and the colony was only 37.68 mm 10 days after inoculation on this medium. The appropriate temperatures for the fungal growth were 5-35℃, but the best growth was observed at 25℃. S. shiraiana could grow on MBDA medium with pH4-11 and the optimal acidity was pH7.0. When saccharide, glucose, sucrose, lactose, maltose, xylose, synanthrin and mannitol were added, respectively, to MBDA as carbon sources, the addition of glucose and mannitol resulted in the best growth. When, were added, respectively, to MBDA as nitrogen sources. Tests of the nitrogen sources showed that the best growth of S. shiraiana was on MBDA plates with the addition of tryptone and the growth was still normal by the addition of other nitrogen sources including potassium nitrate, ammonia nitrate and yeast extract; ammonia phosphate, ammonia sulphate were shown suppressive on growth of the fungus.4. Sensitivity of S. shiriana to fungicides. Seven commonly used fungicides (85% tebuconazole,5% validamycin,25% prochloraz,70% thiophanate-methy,10% difenoconazole,40% dimethachlon and 1% wuyiencin) were tested by adding different concentrations of each fungicide into MBDA medium. Results of the tests showed that the EC50 of the fungicides was 0.0132μg/mL, 0.1516 μg/mL,0.1809 μg/mL,0.2028 μg/mL,0.7867 μg/mL,1.4634 μg/mL and 5.3400 μg/mL, respectively, indicating that 85% tebuconazole was the most suppressive fungicide to S. shiraiana.5. The molecular system of S. shiriana detection. A molecular system based on SCAR-PCR was successfully developed and tested for detection of S. shiraiana in the present study.(1). Selection of specific RAPD primer. A number of 40 random primers were tested using the random amplified polymorphic DNA technique and distinct finger-prints were shown, respectively, by 4 primers. Of the 4 primers, only CS23 (5’-CGCAGCCGAGAT-3’) resulted in 2 specific bands and it was selected as the specific RAPD primer for the detection system.(2). Selection of SCAR primer. Products of the 2 specific bands shown by RAPD primer were collected separately and sequenced. Twenty pairs of primers were designed based on the two specific sequences and one was shown the most idea SCAR primer pair for the PCR amplification of S. shiraiana detection system. This primer-pair was named as SS-F/SS-R. The forward primer was 5’-AGTAAAGAGAACATCAAACTTCG-3’and the reverse primer was 5’-ACTTCCACCGAC CAATCT-3’.(3). Optimization of the detection system. An optimal molecular system was established by adjusting the annealing temperature and concentration of the 4 chemical elements (Mg2+, dNTPs, primer and rTaq enzyme). The constituents of optimized system were:2.5 μL 10×PCR of Buffer, 2.25μLof Mg2+(25 mmol/L),2.5μL of dNTPs(2.5 mmol/L),0.625 μL of SS-F/SS-R (10 μmol/L),1 μL of plate DNA (10 ng/μL), and 0.2 U of rTaq enzyme, adding ddH2O to a total volume of 25μL. The PCR cycle procedures included pre-denaturation at 94℃ for 5 min, denaturing at 94℃for 30 s, annealing at 50℃ for 30 s, extending at 72℃ for 45 s, repeating the procedures for 35 cycles, and supplementary extending at 72℃ for 5 min.(4). Verification of specificity of the detection system. Isolates of 10 related fungal species were tested using the primer SSF/SSR and the optimal PCR detection system. Finger-print was produced and a band of 590bp was clearly shown for all isolates of S. shiraiana, but no band for isolates of the other 9 species of fungi. Namely, only isolates of S. shiraiana were shown positive reactions. Therefore, the SCAR-PCR system established was specific for detecting S. shiraiana only.(5). Sensitivity of the detection system. The original DNA extract (plate) of S. shiraiana was diluted several times by 10-times to get DNA solutions of different concentrations (10 ng,1 ng,100 pg,10 pg,1 pg,100 fg,10 fg and 1 fg/μL). The SCAR-PCR system was applied to amplify the specific DNA sequence from different solutions. Results showed that the specific 590 bp band was present when the DNA solution was diluted to a concentration of 1 pg/μL. This indicated that the PCR detection system was highly sensitive for detecting S. shiraiana.6. Application of the SCAR-PCR detection system. The established SCAR-PCR system was appied to test samples of inoculated mulberry fruits, different field samples and diagnosis of shrunken popcorn disease.(1). Reliability of the detection system. The flowers of mulberry at early anthesis were bagged and were inoculated with mycelium of S. shiraiana when the stigma started to inflate. The fruits from inoculated flowers were sampled 2-5 days (no symptom) and 30 days (diseased) after inoculation and were tested using the established SCAR-PCR system. Results showed that all the inoculated and diseased fruit samples were positive, but the uninoculated samples were negative. These results indicated that the established SCAR-PCR system was 100% reliable for detecting S. shiraiana and it could be applied to detect early infection when the disease symptom was not developed.(2). Application of the SCAR-PCR system to detect S. shiraiana in different mulberry tissues and field soil samples. Two flower samples,3 diseased fruit samples with different symptoms,1 root sample,2 bark samples,2 leaf samples and 12 soil samples were tested. Among all these samples, only the diseased fruit with shrunken symptom was shown positive; all other samples were negative.(3). Disease diagnosis using the SCAR-PCR system. Eight shrunken or diminished and 8 swollen fruit samples were visually distinguished and collected from diseased trees in the field. The SCAR-PCR system was applied to test these samples. The results of the tests showed that 5 of the shrunken or diminished and 1 of the swollen sample were positive; all the other samples were negative. There was obviously a difference between the results of visual and molecular diagnosis. For obvious reason, it was thought that the result from SCAR-PCR diagnosis was more accurate, reliable and practical.To conclude, the shrunken type of popcorn disease on fruit mulberry was caused by S. shiraiana. A molecular SCAR-PCR system using the primer-pair SS-F/SS-R was developed successfully and tested widely with inoculated samples and natural field samples. This detection system was shown highly specific, sensitive, reliable and accurate. It may have great implications in identifying and detecting S. shiraiana, diagnosing and forecasting the popcorn disease.