Identification Of Fungal Pathogens Of Phyllostachys Heteroclada And Analysis Of Disease Occurrence And Development Regularity

Phyllostachys heteroclada Oliv.belongs to Phyllostachys,Bambuseae,Bambusoideae and Poaceae.It is a medium and small monopodium bamboo species which is used in many sorts of ways.Geographically,this kind of bamboo is widely distributed in the Yellow River Basin and in the south,more common in the Sichuan Basin and its marginal mountainous areas.In Sichuan,there are large areas of pure forests in the cities of Ya’an,Yibin and Leshan,and scattered in other regions.It covers an area of around 2×10~4hm~2in Sichuan province,and beyond 0.32×10~4hm~2in Ya’an City.In our daily life,especially in rural areas,this vegetation plays an important role in various objectives,namely ecological protection,landscape architecture,edible production,kinds of implements in various industries and Chinese drugs pharmaceutics etc.In 2015,a comprehensive investigation was performed in different locations where P.heteroclada grows in Ya’an City.As a result,six fungal diseases were discovered,namely rhombic-spot disease,culm rust,two kinds of tarspots,blight and witches-broom.Specially,the rhombic-spot disease of P.heteroclada with some obvious features,such as wide distribution,serious incidence,rapid spread and complex pathogenesis etc,was caused by multiple pathogens.Therefore,these diseases were studied in this thesis,mainly focusing on rhombic-spot disease.And some critical issues were addressed as followed:Taxonomy and identification of causal agents;The occurrence rules,pathogenicity and interaction mechanism of pathogens;Pathogenesis and regularity of disease;Dynamics of microflora and mineral elements from different parts of P.heteroclada during the disease development.The main results of the research are presented as followed:1.Species of pathogen:Based on morphological and multigene phylogenetic analyses,nine pathogenic fungi on P.heteroclada are identified,including two new genera,five new taxa,one new host record species and three new regional record species.The pathogenic microorganisms of rhombic-spot disease are composed of three fungi,namely Neostagonosporella sichuanensis C.L.Yang,X.L.Xu&K.D.Hyde gen.et sp.nov.,Podonectria sichuanensis C.L.Yang&X.L.Xu sp.nov.and Parakarstenia phyllostachydis C.L.Yang,H.O.Baral&X.L.Xu gen.et sp.nov.,which mainly infect culms,twigs and exposed rhizomes;Stereostratum corticioides(Berk.&Broome)H.Magn.,a new regional recorded species,is the causal agent of culm rust on culms and twigs;Phyllachora heterocladae C.L.Yang,X.L.Xu&K.D.Hyde sp.nov.and Bambusicola subthailandica C.L.Yang&Y.G.Liu sp.nov.are the pathogens of tarspot disease.Ph.heterocladae mainly occur on culms and twigs.However,the main injury caused by B.subthailandica is to leaves,and sometimes to culms and twigs;Arthrinium yunnanum D.Q.Dai&K.D.Hyde,mainly infecting on culms and twigs at the top of host,is the pathogen of blight disease and a new record on P.heteroclada;Both of Aciculosporium take I.Miyake and Heteroepichlo?sp.are the new regional recorded species,and could cause witche’s broom on P.heteroclada.In addition,A.yunnanum belongs to a semiparasitic pathogen and may be an opportunistic pathogen,and the rest species are parasitic pathogens(B.subthailandica may be saprophytic).2.The life cycle of pathogen:Through long-term field investigation combined with laboratorial observation and relevant experiments,the living features of Neostagonosporella sichuanensis,Podonectria sichuanensis,Parakarstenia phyllostachydis,Phyllachora heterocladae,Arthrinium yunnanum and Bambusicola subthailandica were clearly summarized,such as the time of occurrence,position of live,type of infected source,way of infection,approach of spread,whether the latent status existed or not and the form of fungi when latent,etc.However,the living habits of Ph.heterocladae,A.yunnanum and B.subthailandica are still incomplete and need to be further observed and improved.3.The diseases of Phyllostachys heteroclada and its relationships:The rhombic-spot disease was the most serious disease and the vital reason resulting in decline and death to the forest of P.heteroclada.The damage caused by the remaining five diseases was far less serious and common than the rhombic-spot disease to P.heteroclada forest.And most of them occasionally occurred with small area of distribution.As mentioned above,Neostagonosporella sichuanensis,Podonectria sichuanensis and Parakarstenia phyllostachydis are the pathogens of the rhombic-spot disease,a successively synergistic effect among them are observed.At the same time,Po.sichuanensis and Pa.phyllostachydis can also cause damage alone,and sometimes grow around the diseased spots caused by other diseases,e.g.tarspot on culms or twigs,which will further aggravate the damage.Culm rust,blight and witches-broom occurred independently,and no synergistic effect among them was observed.However,all of these diseases accelerated the decline and death of P.heteroclada forest when they occurred simultaneously or successively.4.Development and epidemic regularity of rhombic-spot disease:This disease process could be roughly divided into two stages:The first stage is the period of initial infection and spread(November to next April).At first,many brown to dark brown flyspecks were formed on the windward side on the culms and twigs of Phyllostachys heteroclada,and gradually expand to the surrounding areas to form rhombus,sub-rhombus,ellipse or irregular spots.Subsequently,the tissue in the center of the diseased spots raised and generated the hemispherical or subspherical fruiting body which is the ascostroma of Neostagonosporella sichuanensis.These spots were often connnected into a piece or scattered.Ascospores overflowed from ostiole when they matured and then spreaded by the wind,rain or other ways(some scale insects,such as Kuwanaspis phyllostachydis).During this period,N.sichuanensis could generate conidiomata,which were intermixed with ascostromata,or scattered on the diseased tissues.The mature conidia spread together with ascospores.Generally,there is a great outbreak from February to April.Mycelium,ascospore and conidium in the diseased tissues are the main resources of primary infection and invade into the host from the natural wounds and stomatal apparatus or other ways.The second stage is the period of spread and aggravation(May to October).During this time,N.sichuanensis mainly lurk in the form of mycelia and conidia in the diseased tissues,and occasionally generate conidiamata.The mature conidia could spread during this period,but which is not common.In the meanwhile,Podonectria sichuanensis and Parakarstenia phyllostachydis grow continuously around the diseased tissue caused by N.sichuanensis and other pathogens,causing the extension of the diseased spots.The next infection cycle began at November.Synergistic effects,diversity in living habits among pathogens,and susceptibility of the host are the main reasons for the outbreak and prevalence of the disease.Meanwhile,the appropriate temperature and relatively humid environment also contribute to the occurrence of disease.5.Response of microflora to disease:During the development of the disease,the diversity of bambusicolous fungi of Phyllostachys heteroclada was investigated by plate-dilution technique,and the results showed:The bambusicolous fungi were composed of 85.47%acomycetes and 14.52%basidiomycetes,of which ascomycetes included six orders and 21genera,and basidiomycetes included two orders and four genera.Helotiales and Eurotiales were isolated from P.heteroclada for the first time.The variational regularity of Neostagonosporella sichuanensis,Bambusicola subthailandica and Arthrinium yunnanum,which are the pathogens of rhombic-spot disease,tarspot disease and blight disease,respectively,were consistent with the process of disease development.In addition,a variety of potential pathogens were also isolated in our study,such as Penicillium,Fusarium,Didymella,Pestalotiopsis,Cladosporium,Shiraia,Trametes gibbosa and Trametes hirsuta etc,but the role of these fungi to the disease remains to be further studied.The results of bacterial diversity showed that a total of 25 genera and 58 species were isolated,including 17 species of dominant bacteria in 10 genera,namely Bacillus,Brevibacillus,Chryseobacterium,Cupriavidus,Erwinia,Paenibacillus,Pantoea,Pseudomonas,Raoultella and Sphingomonas.There are also potential pathogens among them,e.g.Erwinia rhapontici,but the pathogenic effects on P.heteroclada remains to be studied.Besides,several biocontrol strains were obtained in the experiment,including Trichoderma harzianum,T.koningiopsis,Trichoderma sp.,Clonostachys rosea,Pseudomonas pudita and Paenibacillus illinoisensis,and the efficiency in controling the rhombic-spot disease needs further study and discussion.6.Response of mineral elements to disease:Potassium,calcium and magnesium played an active role in the development of rhombic-spot disease of Phyllostachys heteroclada,while nitrogen was unclear and barely correlated with the disease.There may be a certain synergistic effect between calcium and magnesium,and magnesium was not as protective as potassium and calcium.The ratios of N/Ca,N/Mg,K/Ca and K/Mg of healthy P.heteroclada in various parts were significantly higher than that of the diseased bamboo,and these indexes can be used as one of the indicators for the control effect in the follow-up comprehensive treatment of the disease.