Species Of Lophodermium On Pines In Sichuan And PCR, Cloning And Sequence Analysis Of ITS Regions Of The RRNA Gene

Lophodermiu spp.,causing the falling of pines needles, were investigated thoroughly in pine forests around Sichuan province, including areas in 10 counties and 6 cities. According to the morphological classification method established by Minter, six species of Lophodermium spp., L. conigenum(Brunaud) Hilitz, Lpinastri (Schrad) Chev, L.sichuanense Qiu et Liu, L.indianum Singh et Minter, L.australe Dearn, L.hui (Temporary name) , were found in different areas, different pines(Pinus yunnanensis Franch, P.massoniana Lamb, P. armandi Franch, P.elliottii Engelm, P.tabulaeformis Carr, P.densata Mast) and different parts of pines. The six kind of fungi were isolated after plate culture , and the characteristics of producing spores were studuied. The results showed that Lophodermium spp. cultures could not produce ascocarp in 8 weeks, but in artificial wettish envirnonment the needles with mature ascocarp released ascuspore quickly and the ascuspore germinated soon.By the improving method of benzyl chloride DNA extraction, high -quality DNA from 7 isolates of Lophodermium were obtained. The ITS regions of rRNA gene were amplified by the polymerase chain reaction(PCR) with the fungi general primers ITSt and ITS4. The amplified fragments, about 517bp to 597bp in length, were purified, cloned to pUCm-T vector and transformed into the CaCl2-competent E.coli JM109 host. ITS fragments inserted to pUCm-T vector were sequenced and compared with those in GenBank(Accession number: AY247751,AF473559,AF473555,AY247753). The onious tree was established by the sequence comparison. The results showed that these 11 isolates can be separated into 3 groups.L.conigenum-l,L.conigenum-L.conigenum-3,L.pinastri, AY247751, AF473559, AF473555and AY247753 belong to the first group; L.sichuanense,L.hui belong to the second one and L.conigenum-TM belong to the third one separately. The first group can be divided into two parts, L.conigenum and L.pinastri. In the first group the nucleotide sequencehomology of 5 Lconigenum isolates is above 93.5%, 3 isolates of L.pinastri is above 91.2%, proving that they are different isolates of same species. However the nucleotide sequence homology between L.conigenum and L.pinastri is just about 86.8% -87.5%, indicated inheritance difference between inter-species. L.sichuaneme and L.hui (Temporary name)nucleotide sequence homology is about 90.0%, but it's only 81.0% compared with other Lophodermium spp.. L.conigenum-TM has the lowest homology with others, just about 63.1%-79.3%.The ITS sequences of rRNA gene of L.conigenum-\,L.conigenum-2,L.conigenum-3 and L.pinastri have submitted to Genbank under the accession number AY42248, AY515318, AY515319 and AY422490.