| Phytophthora sojae Kaufmann et Gerdemann is one of the most important plant pathogenic fungus with strong parasites and enormous economic damage on soybean. Based on the isolation of the causal organism of soybean phytophthora root rot in Shandong province, identification of Phytophthora sojae use the method of morphology and polyacrylamide gel electrophoresis of soluble proteins, sequencing of ribosomal DNA-ITS of Phytophthora sojae, effects of medium nutrient on the attachment mode of antheridia on oogonia and on mycelial growth rate of Phytophthora sojae were studied systematically in this dissertation.The main results were as follows.1Isolation and distribution of P. sojae in Shandong provinceThe225soil samples in soybean field were collected from the main soybean producing areas in Shandong province, and Phytophthora sojae was detected by the method of soybean leaf-disc baiting.The result indicated that P. sojae has been baited from16soil samples collected in6counties, including shouguang, rizhao, junan, feixian, zoucheng and jining. A total of5isolates of P. sojae were obtained from soil samples. However, P. sojae has not been detected from81soil samples collected in9cities, including yantai, qingdao, binzhou, zibo, dezhou, jinan, taian, liaocheng and heze.2Identification of Phytophthora sojae use the method of morphology and polyacrylamide gel electrophoresis of soluble proteinsFive isolates of Phytophthora, separated from soybean field in Shandong province, were identified use the method of morphology and polyacrylamide gel electrophoresis of soluble proteins. The results indicated that all of the five tested isolates were Phytophthora, the mycelial growth rate was slow on lima bean agar (LBA) media, hyphae closely, nonseptate, abundant，rounded hyphal swellings in chains, right-angle and shrinking in branch. Sporangium was inverted-pear-shaped, non-papillate, internal proliferation of sporangiophore, zoospores formed within sporangium, homothallism, rounded archegonium and paragynous antheridium. Electrophoresis pattern among the five tested isolates and standard strain were fundamental identical. There were13mutual main bands at the mobility of0.08,0.09,0.11,0.13,0.15,0.18,0.28,0.30,0.32,0.34,0.45,0.52and0.57among five tested isolates and standard strain. The percentage of similarity values among five tested isolates and standard strain were89.66%,100.00%,92.86%,96.30%and92.86%. Based on the morphological characteristies and polyacrylamide gel electrophoresis pattern of soluble proteins, the Five isolates of Phytophthora mentioned above were identified as Phytophthora sojae Kaufmann et Gerdemann3Identification and sequencing of ribosomal DNA-ITS of P. sojaeGenetic polymerase chain reaction（PCR）products from the internal transcribed spacer（ITS） regions of the ribosomal DNA of seven P.sojae isolates （SD145, SD156, SD167, SD191, SD224,7071and AH13）w ere amplified.The results showed that a single band of PCR product about1500bp was amplified from above seven tested isolates.At the meanwhile, the ITS regions of the ribosomal DNA gene from above seven isolates of P. sojae were amplified using the polymerase chain reaction by a couple of specific primers for P. sojae and sequenced. Sequences from the above isolates were compared with published sequences of P. sojae (Accession No.: AF228089, AY277278and FJ801969), P.medicaginis (Accession No.: AF266799), and P.trifolii (Accession No.: AF266800) in Genbank database and phylogenetic trees were produced based on ITS sequence data by “Unweighted Pair-Group Method With Arithmetic Means” mehtods of the software MEGA4and SEAVIEW4. The results indicated that all of the three base sequences of ITS were equal length, and the nucleotide sequence was identical. Three tested isolates of P. sojae (SD145, SD191and AH13), AF228089, AY277278and FJ801969were clustered into the first group, and the sequence of ribosomal DNA-ITS of Shandong isolates were closest relative to that of P. sojae published in GenBank. The two isolates, AF266799(P.medicaginis) and AF266800(P.trifolii) in Genbank, were clustered into another group. The two isolates mentioned above were distant genetic relationship to the other six isolates of P.sojae.4Effeets of medium nutrient on the attachment mode of antheridia on oogonia in P. sojaeThe effects of medium nutrient on the attachment mode of antheridia on oogonia in Phytophthora sojae Kauf. et Gerd were studied.The results showed that the tested isolate SD191formed predominantly paragynous antheridium (PaA) on lima bean agar (LBA), maize granule agar (MA), soybean agar (SBA), carrot agar (CA) and tomato agar (TA) media with94.7%、88.3%、85.0%、84.3%and79.7%paragynous antheridia (paA) respectively, but produced predominantly amphigynous antheridia (AmA) on oat agar (OMA) media (with25.7%AmA). Further investigation indicated that all of the four test isolates origination from different localities formed mostly PaA on LBA (with82.0%to 94.7%of PaA,at the average of87.9%)，b ut produced mostly AmA on OMA(with19.7%to42.3%of PaA, at the average of29.7%), and that the PaA percentage increased with a decrease in OMA concentration (the PaA percentage up to71.0%on1/4strength OMA) and decreased with an decreased in LBA concentration(down to64.3%on1/4strength LBA). The variance analysis showed that PaA percentages among different kinds and concentration of media were significantly different at0.01level. The results mentioned above indicated that the medium nutrient had a significant effect on the expression of the Paragynous and amphigynous characters in Phytophthora sojae Kauf. et Gerd.5Effect of medium nutrient on mycelial growth rate of P. sojaeThe effects of medium nutrient on the mycelial growth rate in P. sojae were studied.The results showed that the mycelial growth rate of the tested isolate SD191on oat agar media (OMA) was the fastesd,which was11.20mm/d；however, the mycelial growth rate of the tested isolate SD191on soybean agar media (SBA) was the slowest,which was1.88mm/d.There was not significant difference on the mycelial growth rate on maize granule agar (MA), lima bean agar (LBA) and tomato agar (TA), with the average rate of9.30mm/d、8.88mm/d and8.22mm/d. Further researched the effect of different media and concentration on the mycelial growth rates of P. sojae.The results indicated that the mycelial growth rate on a high concentration of oat agar media (OMA) was the fastesd, with the average rate of11.20mm/d,but the mycelial growth rate decreased obviously,down to the average rate of6.30mm/d on1/4strength OMA to the contrary, the mycelial growth rate on a high concentration of lima bean agar media (LBA) was slower, with the average rate of8.88mm/d.But the mycelial growth rate increased rapidly with a decrease in LBA concentration, up to the average rate of9.55mm/d on1/4strength LBA. The results mentioned above indicated further that the medium nutrient had a significant effect on the mycelial growth rate of P. sojae... |
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