| Picea is one of the most major edificators consisting of forest in the cold temperate zone.There are about 42 species and 14 varieties around the word.And the Picea which is the main species in subalpine forests is also abundant in China,where 16 species and 9 varieties are cultivated.And there are 11 species and 2 varieties of Picea in Sichuan province.As we all know,subalpine coniferous forests in west Sichuan form the ecological barrier of the upper Yangtze River,and spruce is the main composition.In the 1960s,spruce is chosen as a good pioneer for forestation,and then a large number of pure forests are planted in west Sichuan.In the end of 1990s,there are about 33333 hm2 diseased spruce forests among the 266666 hm2 spruce plantations.At present,spruce in plantation forest of Sichuan is heavily diseased.According to reports,there are three needle diseases that could cause the needle cast all over the world caused by Lophodermium piceae,Rhizosphaera kalkhoffii,Chrysomyxa spp..In China,the studies on the pathogens of the needle disease are still lacking,most of the researches are about the control techniques on this disease.In this paper,surveys are conducted in 13 planted spruce forests from Luding county,Pingwu county,Tianquan county and Songpan county,et al..and we study the main needle disease in different spruce forests.We isolate the needle endophytic fungi from needle to analyze the species and ecological status of pathogens and potential pathogens on spruce.With the aid of optical microscope and scanning electron microscope,the morphological characters of the pathogens are described.In addition,the phylogeny is analyzed.Finally,we get the following results:1.Lophodermium piceae,Rhizosphaera kalkhoffi,Chrysomyxa spp.are the pathogens that infect the spruce in spruce plantation forest,Sichuan.Finally,61 species of endophytic fungi belonging to 37 genera are isolated from spruce needle.Twenty species from 16 genera are isolated from yangdonghe,Pingwu county.Aspergillus fumigates,Rhizosphaera kalkhoffii and Cladosporium cladosporioides are the predominant strains that have more than 50%isolation frequency.12 species from 9 genera are isolated from Machangliang.Thielavia microspora,Phoma sp.,R.kalkhoffii and C.cladosporioides are the dominant species.5 species of 5 genera are isolated from Erlangshan,and R.kalkhoffii is the dominant species on both needles.21 species from 14 genera are isolated from Baishahe,three species,Schizophyllum commune,Stagonosporopsis cucurbitacearum and Aspergillus fumigates are the dominant species.But on the healthy spruce from Xiaoxing,several Penicillium strains are the dominant species.R.kalkhoffii is the common predominant strains in three forests.Finally,there are five potential pathogens on spruce are isolated,Sydowia polyspora,Phoma sp.,Phomopsis sp.,Mycosphaerella sp.,Schizophyllum commune.2.The pathogen R.kalkhoffii is isolated from needle with high separation frequency at Yangdonghe,Erlangshan and Machangliang.Firstly,this pathogen is easily isolated as endophytic fungi in spring.The isolation frequency on 2-year-old needle is 58.82%,and 85.52%on one year old needle.And it is also isolated from the fallen needle at Yangdonghe and Erlangshan.It follows that R.kalkhoffii can live on the alive needle and dead needle in large area.There are two type of conidium produced on the PDA medium.The size of the small ones are 5.3?10 X 3.3?5.8?m.And the larger ones are septate with diaphragm of one to three,11.2?19.0 ?mx5.0?8.5 ?m,but two celled conidium is more common.The germ tubes usually happen on the both ends of conidium,seldom from the separate.According to the reports in the literature,this fungus produces fruit body on the dead needle to overwinter,and then infect the new needle.But no fruit body is found on the alive needle even on dead needle.This fungus lives in needles in the form of hyphae.3.There are variations in the morphology and ITS sequences among L.piceae.Like the L.piceae from Picea abies and L.nanakii,the ascoma hump moderately,with no lip cell,but the ascoma,ascusis,ascospore from our plots are larger than them.In addition,there are some differences in the size of ascoma.ascus and ascospore among our sampling plots.The L.piceae in our plots is highly similar to those on P.abies according the blast in NCBI.And the ITS-rDNA of the strains have high diversity even if they come from the same geographical location.The genetic difference has nothing to do with the geographical location as showed in the phylogenetic tree.Because of the absent record on L.nanakii in GenBank,sequence analysis between them failured.4.A new species of Lirula is found on picea batfouriana based on morphology and molecular study directly from ascoma.This pathogen infects the two-year old needles and results in death.And it is the first report of spruce needle blight disease in west Sichuan.Lirula is distributed in North America,Europe and Asia,and has strong parasitic ability that infect Picea and Abies.There are 11 species and one varieties in the genus,and 3 species are reported on Picea.This fungus appears on the two-year-old needles,and the ascoma mature at the end of April when the current year needles don't bud.The ascospore mainly infects the one-year-old needle,and then diseased needles wither during the following time.At present,there are three species of Lirula on Picea,namely L.brevispora,L.macrospora and L.filiformis.But there are many differences between the isolates and those three species.But the similarity with L.macrospora is 96%based on ITS-rDNA.Moreover,this new species is strongly parasitic and pathogenicity on spruce,and has a tendency to spread.We should strengthen the monitoring and control.5.In our study,the needle rust is caused by five species of genus Chrysomyxa.With the help of light microscope and electron microscope,five different Chrysomyxa species are found in four spruce forests,including a microcyclic on the two-year old needles.According to the surface structure of the aecidiospore,the warts are divided into three types.The surface structure of aecidiospores on both P.likiangensis and P.likiangensis var.linzhiensis are same,as well as the size of aecidiospores.In addition,the results of blastn between the two rusts show that the ITS sequence similarity is 100%..However,the rusts from other forests have other types of warts with a base composed of filament on the surface of aecidiospores.In addition,the length of the warts and the size of aecidiospores are obviously different.All the aecidiospores have no smooth cap or longitudinal groove compared with the species reported abroad.Chrysomyxa,which is the pathogen of needle rust,bud rust and cone rust on spruce,has heteroecism.This fungus alternate on Rhododendroideae,Empetraceae,Pyroloideae,Vaccinioideae.Two type of telium,cushion-like and head-shape coincide on Rhododendron in plots.The head-shape telium is unique to Asia.The phylogenetic analysis based on ITS and 28S rDNA between aecidiospore on spruce and telium on alternate hosts show close relationships.Moreover,the head-shaped teliums from three plots are divided into three clades in the phylogenetic tree,which further illustrates the different species of Chrysomyxa in west China.The phylogenetic analysis of teleomorphic and anamorphic states could help us to understand the life cycle of Chrysomyxa.6.Seven genes are amplified on Chrysomyxa.Finally,ribosomal gene(ITS-rDNA,28S Rdna,18S rDNA)are better than the mitochondrial gene(CO1,C03,NAD 6,ATP 6),which have high stability to amplify successfully.In this paper,we mainly use the 28S rDNA to build the phylogenetic tree on Chrysomyxa,which could also reflect the differences among the Chrysomyxa species.Blastn between telium and aecidiospore based on ITS rDNA show higher variability than on 28S rDNA,which may result from the conservation of 28S(large subunit)and variability of ITS.Once enlarge the data,Chrysomyxa species are easily confused with other rust genus in the phylogenetic tree based on ITS rDNA,such as Cronatium sp.,Coleosporium sp.,Melampsoridium sp.Due the conservation on SSU,the sequencing results show that the Chrysomyxa is highly similarity with the other rust genus that they consistency can reach 99%?100%.Therefore,different rust genus could not be clustered based on the sequence of SSU. |
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