Species-specific PCR-based assays for identification and detection of Botryosphaeriaceae species causing stem blight on blueberry in China

Botryosphaeriaceae species are important causal agents of blueberry stem blight worldwide. Blueberry stem blight has become an important disease, potentially affecting the quality and production of blueberries in China. It is difficult and time-consuming to identify at the species level using morphological methods. The aim of this study was to develop polymerase chain reaction（PCR） assays for the diagnosis and early detection of latent infections of blueberry stems by Botryosphaeria spp. Species-specific primers, based on the ribosomal DNA internal transcribed spacer region and β-tubulin gene, were designed and selected for use in PCR assays. Three primer pairs, Lt347-F/R for Lasiodiplodia theobromae, Np304-F/R for Neofusicoccum parvum and FaF/Bt2b for Botryosphaeria dothidea, successfully amplified specific PCR fragments of different sizes on pure cultures or from blueberry stems inoculated and naturally infected blueberry plants with three pathogens, respectively. These primers did not amplify any PCR fragments from other blueberry stem disease-associated pathogens, such as Phomopsis spp. and Pestalotiopsis spp. This PCR protocol could detect as low as 1 00 pg to 1 ng of purified fungal DNA. This PCR-based protocol could be used for the diagnosis and detection of these pathogens from pure cultures or from infected blueberry plants.

Species of Botryosphaeriaceae involved in grapevine dieback in China

Botryosphaeria dieback is a serious disease problem for table and grape wine production worldwide.The disease however,has been less well-studied in China.In this study,we surveyed Botryosphaeria dieback in 72 vineyards of 20 grape-growing regions in China and found that Botryosphaeria dieback occurs in 18 out of 20 provinces.Morphological and multi-gene phylogenetic analyses confirmed that Botryosphaeria dothidea,Diplodia seriata,Lasiodiplodia theobromae and Neofusicoccum parvum are associated with different grapevine dieback symptoms.This study also revealed considerable differences in the geographical distribution of Botryosphaeriaceae species in China with Lasiodiplodia theobromae and Neofusicoccum parvum occurring only in subtropical monsoon climate regions,Diplodia seriata occurring only in temperate monsoon climate regions,and Botryosphaeria dothidea occurring in both temperate and subtropical monsoon climate regions.Analysis of 26 isolates showed that there is little genetic variation within species.Koch’s postulates were satisfied for all species,and pathogenicity testing showed that among the 25 major cultivars growing in China,none was resistant to the four taxa.The current paper represents the first detailed report on Botryosphaeria dieback of grapevines in China.

Lasiodiplodia pseudotheobromae causes stem canker of Chinese hackberry in China

Chinese hackberry(Celtis sinensis Pers.)is an adaptable species widely growing in southern China.The symptoms of canker on stems of seedlings were discovered mid-July 2017 in Shuyang,Jiangsu Province.The diseased portions of the stems were dark brown due to discoloured xylem.Some seedlings showed symptoms of wilting,leaf fall,twig dieback,and tissue discolouration.The outbreak period was concentrated in July and August,suggesting that the disease spread during summer months.Possible fungal causal agents were isolated from naturally infected canker tissue and discoloured xylem.The isolate from xylem tissue with a high frequency(>50%)was named Ls7 type.Pathogenicity tests were carried out on 4-yearold seedlings.The symptoms of canker began to develop 20 days after inoculation with Ls7 isolate and by day 35,there were dark,enlarged longitudinal lesions.A phylogenetic tree of the isolate was developed using the internal transcribed spacer,elongation factor-1α(tef1-α),β-tubulin gene(TUB)and RNA polymerase II subunit primer genes(RPB2).Based on morphological features and phylogenetic information,the pathogen was identifi ed as Lasiodiplodia pseudotheobromae.This is the fi rst report of L.pseudotheobromae causing canker on Chinese hackberry stems in China.

Multiple locus genealogies and phenotypic characters reappraise the causal agents of apple ring rot in China

Apple ring rot inflicts severe economic losses in the main apple producing areas of East Asia.The causal agent of the disease has been variously identified as Macrophoma kuwatsukai,Physalospora piricola and Botryosphaeria berengeriana f.sp.piricola,although B.dothidea is currently the most widely accepted pathogen name.The taxonomic uncertainty has delayed research that is needed to manage effectively this destructive disease.In the present study,genealogical concordance phylogenetic species recognition(GCPSR)was applied to pathogenic fungal isolates from apple and pear from several locations in China,along with several reference isolates.Phylogenetic results based on sequences of four nuclear loci(ITS,EF-1α,HIS and HSP)revealed the existence of two species within the examined isolates.One includes an ex-epitype isolate of B.dothidea and the other includes an isolate that was previously designated as B.berengeriana f.sp.piricola.Morphologically,the latter taxon presented an appressed mycelial mat on PDA whereas B.dothidea displayed columns of aerial mycelia reaching the lids,and conidia of the latter species were longer than B.dothidea.Botryosphaeria dothidea had a faster growth rate than the latter taxon under relatively high temperatures.Pathogenicity tests showed that on pear stems the latter taxon caused large-scale cankers along with blisters whereas B.dothidea was non-pathogenic,but on apple shoots the two fungi induced large and small wart-like prominences,respectively.Overall,this cryptic species demonstrated sufficient genetic variations and biological differences from B.dothidea.As a result of taxonomic study,we described here the latter taxon in a new combination,Botryosphaeria kuwatsukai and designate an epitype.Both B.kuwatsukai and B.dothidea are considered to be the main causal agents for apple ring rot in China and Japan.

Towards a natural classification of Botryosphaeriales

The type specimens of Auerswaldia,Auerswaldiella,Barriopsis,Botryosphaeria,Leptoguignardia,Melanops,Neodeightonia,Phaeobotryon,Phaeobotryosphaeria,Phyllachorella,Pyrenostigme,Saccharata,Sivanesania,Spencermartinsia and Vestergrenia were examined and fresh specimens of Botryosphaeriales were collected from Thailand.This material is used to provide a systematic treatment of Botryosphaeriales based on morphology and phylogeny.Two new genera,Botryobambusa and Cophinforma are introduced and comparedwith existing genera.Four species newto science,Auerswaldia dothiorella,A.lignicola,Botryosphaeria fusispora and Phaeobotryosphaeria eucalypti,are also described and justified.We accept 29 genera in Botryosphaeriales,with Macrovalsaria being newly placed.In the phylogenetic tree,the 114 strains of Botyrosphaeriales included in the analysis cluster into two major clades with 80%,96%and 1.00(MP,ML and BY)support,with Clade A containing the family type of Botryosphaeriaceae,and Clade B containing Phyllosticta,Saccharata and Melanops species.This group may represent Phyllostictaceae.In Clade A the taxa analyzed cluster in eight sub-clades(Clades A1-8).Clade A1 comprises three distinct subclusters corresponding to the genera Diplodia(Diplodia Clade),Neodeightonia(Neodeightonia Clade)and Lasiodiplodia(Lasiodiplodia Clade).Clade A2 clusters into three groups representing Phaeobotryosphaeria(100%),Phaeobotryon(100%)and Barriopsis(94%).Clade A3 incorporates 17 strains that cluster into three well-supported genera(Dothiorella(86%),Spencermartinsia(100%)and Auerswaldia(63%);the position of Macrophomina is not stable.Clade A4 is a single lineage(100%)representing the new genus Botryobambusa.Clade A5 is a wellsupported subclade incorporating Neofussicoccum.Clade A6 represents the type species of Botryosphaeria,three other Botryosphaeria species and two other genera,Neoscytalidium and Cophinforma gen.nov.Clade A7 comprises two Pseudofusicoccum species and Clade A8 has two Aplosporella species.These sub-clades may eventually require separate families but this requires analysis of a much larger dataset.Our data advances the understanding of Botryosphaeriales,there is,however,still much research to be carried out with resolution of families and genera,linkage of sexual and asexual morphs and differentiation of cryptic species.

Bambusicolous fungi

Fourty-three species of microfungi from bamboo are treated,including one new family,Occultibambusaceae,three new genera,Neoanthostomella,Occultibambusa and Seriascoma,27 new species,one renamed species and 15 redescribed or re-illustrated species,and four designated reference specimens are treated in this paper,the majority of which are saprobic on dead culms.To determine species identification,separate phylogenetical analyses for each group are carried out,based on molecular data from this study and sequences downloaded from GenBank.Morphologically similar species and phylogenetically close taxa are compared and discussed.In addition a list of bambusicolous fungi published since Hyde and colleagues in 2002 is provided.

Species of Lasiodiplodia associated with papaya stem-end rot in Brazil

This study aims to identify and characterize species of Lasiodiplodia associated with stem-end rot of papaya in six different populations in the Northeast of Brazil.Fungal identifications were made using a combination of morphology together with a phylogenetic analysis based on partial translation elongation factor 1-αsequence(EF-1α)and internal transcribed spacers(ITS).Five species of Lasiodiplodia were identified:Lasiodiplodia brasiliense sp.nov.,L.hormozganensis,L.marypalme sp.nov.,L.pseudotheobromae and L.theobromae.Only L.theobromae had previously been reported in papaya,while all the other species are reported for the first time in association with this host in Brazil and worldwide.Lasiodiplodia theobromae was the most prevalent species.All species of Lasiodiplodia were pathogenic on papaya fruit,with L.hormozganensis being the most virulent.

Botryosphaeria, Neofusicoccum, Neoscytalidium and Pseudofusicoccum species associated with mango in Brazil

Members of the Botryosphaeriaceae are well known fungi associated with dieback,canker and fruit rot on various hosts worldwide,including mango.The aim of this study was identify a large collection of Botryosphaeriaceae species associated with dieback and stem-end rot of mango in the semi-arid region of Northeastern Brazil,and compare the species in relation to mycelial growth,pathogenicity and virulence.A total of 115 isolates were sampled and based on morphology and DNA sequence data(ITS and EF1-α)seven taxa were identified,namely,Botryosphaeria dothidea,B.mamane,Fusicoccum fabicercianum,Neofusicoccum parvum,N.brasiliense sp.nov,Neoscytalidium dimidiatum and Pseudofusicoccum stromaticum.B.dothidea and P.stromaticum were the most commonly isolated species,which represented 37%and 33%of all isolates respectively.B.mamane is reported for the first time in association with mango diseases worldwide.There were significant differences among the species obtained in this study in relation to optimum temperature for mycelial growth and mycelial growth rates.All species were pathogenic on mango fruit.There were significant differences in virulence among the species,with Ne.dimidiatum and N.parvum being the most virulent species,while P.stromaticum was the least virulent.

Species of Lasiodiplodia associated with mango in Brazil

Mango(Mangifera indica)is a major tropical fruit species cultivated in Brazil.The objective of this study was to identify species of Lasiodiplodia associated with dieback and stem-end rot of mango in the semi-arid region of Northeastern Brazil,and compare the species in relation to mycelial growth,pathogenicity and virulence.A total of 120 isolates of Lasiodiplodia were used and identifications were made using a combination of morphology and phylogenetic analysis based on partial translation elongation factor 1-αsequence(EF1-α)and internal transcribed spacers(ITS).The following species were identified:Lasiodiplodia crassispora,L.egyptiacae,L.hormozganensis,L.iraniensis,L.pseudotheobromae,L.theobromae and Lasiodiplodia sp..Lasiodiplodia theobromae was the most frequently isolated species,which represented 41%of all the isolates.Only this species had been previously reported on mango in Brazil,while the other species represent the first report associated with mango tree diseases in this country.Lasiodiplodia crassispora is reported for the first time associated with mango diseases worldwide.There were significant differences in mycelial growth rates among the Lasiodiplodia species and also in the optimum temperature for growth.All species of Lasiodiplodia were pathogenic on mango fruit.There were significant differences in virulence among the species,wherein L.hormozganensis and Lasiodiplodia sp.were the most virulent,while the least virulent were L.iraniensis,L.pseudotheobromae,L.crassispora and L.egyptiacae.

The complex of Diplodia species associated with Fraxinus and some other woody hosts in Italy and Portugal

Studies on the taxonomy and phylogeny of Diplodia have been hampered by the lack of an ex-type culture linked to the holotype of D.mutila,which is the type of the genus.In this study a large collection of Diplodia strains,obtained from ash and other woody hosts showing V-shaped cankers and branch dieback,were identified based on morphological characters and DNA sequence data from ITS and EF1-α loci.Results of combined morphological and phylogenetic analyses showed that the Fraxinus isolates from Italy,the Netherlands,Portugal and Spain belong to three distinct species namely Diplodia fraxini,Diplodia mutila and Diplodia subglobosa sp.nov.An epitype was designated for Diplodia mutila,with associated ex-epitype cultures.The name D.fraxini is re-instated and a neotype designated.Two species,Diplodia seriata and Diplodia pseudoseriata were reported for the first time on Fraxinus spp.

Characterization of fungal pathogens associated with grapevine trunk diseases in Arkansas and Missouri

Grapevine trunk diseases are a major concern to the wine-and table-grape industries worldwide,limiting both vineyard longevity and productivity.Field surveys conducted throughout the grape-growing regions of Arkansas and Missouri revealed the presence of three economically important grapevine trunk diseases including,Botryosphaeria canker,Eutypa dieback and esca.Morphological studies along with multi-gene phylogenetical analyses confirmed the identification of 15 different fungal taxa associated with different vascular symptoms.These include Botryosphaeria dothidea,Diatrypella sp.,Diplodia seriata,Dothiorella americana,Eutypa lata,Eutypella vitis,Lasiodiplodia missouriana,Lasiodiplodia viticola,Neofusicoccum ribis,Neofusicoccum vitifusiforme,Pestalotiopsis sp.,Pestalotiopsis uvicola,Phaeomoniella chlamydospora,Phomopsis viticola,Schyzophyllum commune,and Togninia minima.All of these represent new records on grapevines in Arkansas and Missouri.Dothiorella americana,L.missouriana and L.viticola are described as new species,and both N.ribis and N.vitifusiforme are first reported as grapevine pathogens in North America.Koch’s postulates confirmed the pathogenicity of all fungal species except S.commune in the interspecific hybrids Vignoles,Chambourcin,Norton,and Traminette.Lasiodiplodia spp.,N.ribis,and P.viticola were the most virulent fungi,while D.americana,E.vitis and N.vitifusiforme were considered to be weak pathogens.This research highlights the importance that grapevine trunk diseases have on grapevine health in growing regions where,due to different climatological conditions,interspecific hybrid cultivars are predominantly grown.

Dothiorella vidmadera, a novel species from grapevines in Australia and notes on Spencermartinsia

Strains of a coelomycete isolated from grapevine cankers in southeastern Australia and identified as Dothiorella iberica in previous studies are redescribed in this study as a novel species based on morphological characters and phylogenetic analyses of DNA sequences of the internal transcribed spacer region(ITS1-5.8S-ITS2),and partial sequences of the translation elongation factor 1-αandβ-tubulin genes.Dothiorella vidmadera sp.nov.is most closely related to D.iberica,D.americana and D.sarmentorum,but differs in morphological characters and DNA sequences.All four species are known to be associated with Botryosphaeria dieback of grapevines.Additionally,an unidentified ascomycete isolated from dead wood of grapevines in Western Australia is described.Phylogenetically,strains were most closely related to Spencermartinsia viticola,and bore conidia with morphological characters and dimensions consistent with published descriptions.However,ascospores were shorter and narrower than previously reported and lacked the terminal apiculi that typify the genus.