Identification And Phylogenetic Analysis Of Four Fungal Genera Isolates Associated With Diseased Litchi Fruits In China

Litchi(Litchi chinensis Sonn.)is one of the famous fruits in southern China.Litchi fruiting period happens to be at the hot and rainy season,which facilitates microbial growth and fruit infection.Diseases have become an important factor in reducing production and worsening the quality of litchi.Accurate identification of pathogens is the basis and precondition for effective disease control.However,previous identification of litchi pathogens in China was mostly based on morphological characteristics,which might sometimes lead to confusion on species identification of pathogens,because the morphological characteristics might change under different environmental conditions.This is not conducive to disease monitor and control,quarantine management,plant breeding of resistant cultivars,and so on.Recently,phylogenetic analysis has been extensively used in identification of plant pathogens.The species concept and taxonomic status of many important plant pathogenic fungi have also undergone major changes.In this study we systematically collected fungal isolates from diseased fruits of litchi,with special focus on the isolates belonging to the genera of Colletotrichum,Botryosphearia sensu lato,Pestalotiopsis sensu lato and Peronophythora.One hundred and seventy-one of these isolates were identified based on morphological and multi-gene phylogenetic analyses,and the results are as follows:1.Identification of Colletotrichum isolates associated with diseased litchi fruits: By coupling morphological characteristics and combined sequence data of internal transcribed spacer(ITS),β-tubulin(TUB),actin(ACT),glyceraldehyde-3-phosphate dehydrogenase(GAPDH)and glutamine synthetase(GS)gene regions,we showed that the 110 Colletotrichum isolates belonged to the Colletotrichum acutatum species complex,Colletotrichum boninense species complex,Colletotrichum gloeosporioides species complex and Colletotrichum orchidearum species complex,respectively,including 5known species,i.e.,Colletotrichum fioriniae,Colletotrichum fructicola,Colletotrichum karsti,Colletotrichum plurivorum and Colletotrichum siamense.Among them,there were69 Colletotrichum siamense isolates and 34 Colletotrichum fructicola isolates,accounting for 62.7% and 30.9% of the total Colletotrichum isolates,respectively.Colletotrichum siamense and Colletotrichum fructicola,which both belonged to the Colletotrichum gloeosporioides species complex,together accounted for up to 93.6% of the Colletotrichum isolates.Colletotrichum gloeosporioides sensu stricto was not found in this study.We concluded that Colletotrichum gloeosporioides sensu stricto is not a common pathogen of litchi anthracnose in mainland China.2.Identification of Botryosphearia sensu lato isolates associated with diseased litchi fruits: By coupling morphological characteristics and combined sequence data of ITS,TUB and translation elongation factor(TEF)gene regions,we showed that the 36 Botryosphearia sensu lato isolates belonged to the Lasiodiplodia spp.,Neofusicoccum spp.,Botryosphaeria sensu stricto spp.and Cophinforma spp.,including 10 known species and 2undefined species,which were Botryosphaeria dothidea,Botryosphaeria fusispora,Cophinforma atrovirens,Lasiodiplodia citricola,Lasiodiplodia gonubiensis,Lasiodiplodia hormozganensis,Lasiodiplodia pandanicola,Lasiodiplodia theobromae,Neofusicoccum parvum,Neofusicoccum sinoeucalypti,Botryosphaeria sp.and Lasiodiplodia sp.respectively.Among them,the most common species was Botryosphaeria dothidea with 9isolates,accounting for 25.0% of the total Botryosphearia sensu lato isolates.3.Identification of Pestalotiopsis sensu lato isolates associated with diseased litchi fruits: By coupling morphological characteristics and combined sequence data of ITS,TUB and TEF gene regions,we showed that the 13 Pestalotiopsis sensu lato isolates belonged to Pestalotiopsis sensu stricto spp.and Neopestalotiopsis spp.respectively,including 4 known species and 6 undefined species,which were Neopestalotiopsis cubana,Neopestalotiopsis protearum,Neopestalotiopsis surinamensis,Pestalotiopsis trachicarpicola,Neopestalotiopsis sp.1,Neopestalotiopsis sp.2,Pestalotiopsis sp.1,Pestalotiopsis sp.2,Pestalotiopsis sp.3 and Pestalotiopsis sp.4,without significantly dominant species being found.4.Identification of Peronophythora isolates associated with diseased litchi fruits: By coupling morphological characteristics and combined sequence data of ITS,TUB and TEF gene regions,we showed that the 12 Peronophythora isolates from different provinces in China were clustered with the Peronophythora litchii reference strains forming a monophylogenetic group.Therefore,we concluded that all the 12 Peronophythora isolates belonged to Peronophythora litchii with no further differentiation.In summary,this study further clarified the species composition of Colletotrichum,Botryosphearia sensu lato,Pestalotiopsis sensu lato and Peronophythora associated with diseased litchi fruits,which substantially enriches the etiology study of litchi diseases.The findings from this study could be useful clues for identification of the pathogens of litchi diseases and for effective prevention and control of diseases.At the same time,the results might also provide a good reference for breeding of litchi disease resistant cultivars and for custom phytosanitary.