Identification of Fungal Species Associated with Apple Canker in Tarim Basin, China.

Apple canker has decreased yields of the economically important apple (Malus domestica) crop in China in recent years. Pathogen identity is highly challenging and the disease is poorly understood. Specimens of 339 fungi were isolated from apple trees in the primary apple-producing region in the Tarim Basin during the current study. In total, nine species of Cytospora and five species of the family Botryosphaeriaceae were identified by morphological observation and multilocus phylogenetic analyses: internal transcribed spacer (ITS), actin, translation elongation factor (TEF), and ß-tubulin (TUB) gene regions for Cytospora and ITS, TEF, and TUB for Botryosphaeriaceae. Cytospora pyri from the Cytospora genus was the dominant species causing apple canker in the Tarim Basin. C. melnikii, C. tritici, C. euonymina, Diplodia seriata, and Botryosphaeria dothidea have been described as the cause of apple canker in China. Apple (Red Fuji) branches were utilized to assess the pathogenicity of 24 representative fungal isolates from the 14 species, and branches from seven distinct woody plants (Korla pear, walnut, Chinese date, Xinjiang poplar, sand jujube, Populus euphratica, and willow) were utilized to analyze the host range. The main pathogenic fungal species of apple canker around the Tarim Basin were identified and biological characteristics explored. Pathogen diversity and regional source diversity were assessed with host range and pathogenicity. The aim was to provide a theoretical foundation for the prevention and treatment of apple canker.

First report of Diplodia mutila associated with Botryosphaeria canker and dieback of apple trees in Xinjiang, China.

Apple (Malus pumila Mill.) is an important fruit crop in Xinjiang, China. In September 2021, apple tree canker was observed in a 21-year-old commercial apple orchard cv. Fuji in Xinjiang (38°17'51.43"N, 77°9'50.81"E) , northwest of China. Of the 200 plants surveyed, 25% were symptomatic. The diseased trees showed branch dieback and cankers. The cankers observed on the wood were sunken, shriveled, and discolored. After the bark was peeled off, the diseased wood was dark brown, and the necrosis was obvious on the cross-section of the diseased branch. To identify the causal agent, five symptomatic trees were collected and analyzed in the laboratory. Apple wood samples (0.5×0.5 cm) were surface-disinfected with 1% v/v sodium hypochlorite and 75% v/v ethanol, rinsed with sterile distilled water, transferred onto potato dextrose agar (PDA), and incubated in the dark at 25 °C for 5 days. Conidia were induced on sterilized pine needles covered with 2% w/v water agar under near-UV light. The colonies of five isolates were white to gray with sparse aerial mycelium that gradually became dark olive green in the later stage. Conidia were initially hyaline but becoming brown at maturity, 1-septate, oval, rounded at both ends, and with dimensions of 24.9-32.1 × 15.1-21.5 µm (n =50) and the aspect ratio of 1.6. Based on the cultural and morphological features of Phillips (2002), the isolates were identified initially as Diplodia mutila (Fr. : Fr.) Mont. To confirm species identification, genomic DNA was extracted from the representative isolate SC-8A. The primer ITS1/ITS4, EF1-728F/EF1-986R and BT2a/BT2b were used to amplify the rDNA sequences of, respectively, the internal transcribed spacer (ITS), translation elongation factor 1-alpha (EF1-α) gene, and a portion of beta-tubulin (tub2) gene. The nucleotide sequences indicated ≥99% identity to D. mutila (CBS 112553) for three DNA regions. Consensus sequences were deposited in GenBank. as accession numbers OM618108, OM676657 and OM676658 for ITS, EF1-α and tub2, respectively. To fulfill Koch's postulates, pathogenicity tests were performed using isolate SC-8A on one year old branches of cv. Fuji (n=5). Wounds were created in the middle of the branches using a sterilized hole punch (5mm diameter) and were immediately inoculated with mycelial plugs of the same diameter. For the control treatment, sterile agar plugs were used (n=5) in the branches. The inoculated and control branches were wrapped with sterile parafilm. On the 10th day after inoculation, canker lesions appeared on the inoculated branches, but no lesions were observed in the negative control. D. mutila was re-isolated from 100% of the inoculated shoots and was not re-isolated from any of the negative controls, the Koch's postulates were met. Previously, D. mutila has been reported in Canada (Úrbez-Torres et al., 2016), Argentina (Lódolo et al., 2022) and Chile (Díaz et al., 2022) causing Botryosphaeria canker and dieback in apples. To our knowledge, this is the first report of D. mutila causing Botryosphaeria canker and dieback in apple trees in China.

Assessment of diversity and genetic relationships of Neonectria ditissima: the causal agent of fruit tree canker.

BACKGROUND: Neonectria ditissima is one of the most important fungal pathogens of apple trees, where it causes fruit tree canker. Information about the amount and partitioning of genetic variation of this fungus could be helpful for improving orchard management strategies and for breeding apple cultivars with high levels of genetically determined resistance. In this study single-spore Neonectria isolates originating from both the same and from different perithecia, apple cultivars and apple orchards in Sweden and Belgium, were evaluated for AFLP- and SSR-based genetic similarity and for mating system. RESULTS: Seven SSR loci produced a total of 31 alleles with an average of 4 alleles per locus, while 11 AFLP primer combinations produced an average of 35 fragments per primer combination and 71 % polymorphic fragments. An AFLP-based analysis of molecular variance (AMOVA) revealed that 89 % of the variation was found within orchards and 11 % between orchards. Genetic similarity among the studied isolates was illustrated with a principal coordinate analyseis (PCoA) and a dendrogram. AFLP-based Jaccard's similarity coefficients were the highest when single-ascospore isolates obtained from the same perithecium were compared, medium-high for isolates from different perithecia on the same tree, and lowest when isolates from different trees were compared. CONCLUSIONS: Based on the results of PCoA and AMOVA analysis, isolates from the same or geographically close orchards did not group together. Since AFLP profiles differed also when single-ascospore isolates from the same perithecium were compared, the mating system of N. ditissima is most likely heterothallic.

Comparative genomics of five Valsa species gives insights on their pathogenicity evolution.

Valsa is a genus of ascomycetes within the Valsaceae family. This family includes many wood destructive pathogens such as the well known Valsa mali and Valsa pyri which cause canker diseases in fruit trees and threaten the global fruit production. Lack of genomic information of this family is impeding our understandings about their evolution and genetic basis of their pathogenicity divergence. Here, we report genome assemblies of Valsa malicola, Valsa persoonii, and Valsa sordida which represent close relatives of Valsa mali and Valsa pyri with different host preferences. Comparative genomics analysis revealed that segmental rearrangements, inversions, and translocations frequently occurred among Valsa spp. genomes. Gene families that exhibited gene copy expansions tended to be associated with secondary metabolism, transmembrane transport, and pyrophosphatase activities. Orthologous genes in regions lost synteny exhibited significantly higher rate of synonymous substitution (KS) than those in regions retained synteny. Moreover, among these genes, membrane transporter families associated with antidrug (MFS, DHA) activities and nutrient transportation (SP and APCs) activities were significantly over-represented. Lineage specific synonymous substitution (KS) and nonsynonymous substitution (KA) analysis based on the phylogeny constructed from 11 fungal species identified a set of genes with selection signatures in Valsa clade and these genes were significantly enriched in functions associated with fatty acid beta-oxidation, DNA helicase activity, and ATPase activity. Furthermore, unique genes that possessed or retained by each of the five Valsa species are more likely part of the secondary metabolic (SM) gene clusters. SM gene clusters conserved across five Valsa species showed various degrees of diversification in both identity and completeness. All 11 syntenically conserved SM clusters showed differential expression during the infection of apple branch with Valsa mali suggesting involvements of secondary metabolism in the pathogenicity of Valsa species.

Identification and Characterization of Pseudomonas syringae pv. syringae, a Causative Bacterium of Apple Canker in Korea.

In the present investigation, bacterial isolates from infected apple trees causing apple canker during winter were studied in the northern Gyeongbuk Province, Korea. The pathogen was identified as Pseudomonas syringae pv. syringae (Pss) through various physiological and biochemical characterization assays such as BIOLOG, gas chromatography of fatty acid methyl esters, and 16S rRNA. Bioassays for the production of phytotoxins were positive for syringopeptin and syringomycin against Bacillus megaterium and Geotrichum candidum, respectively. The polymerase chain reaction (PCR) method enabled the detection of toxin-producing genes, syrB1, and sypB in Pss. The differentiation of strains was performed using LOPAT and GATTa tests. Pss further exhibited ice nucleation activity (INA) at a temperature of -0.7°C, indicating an INA+ bacterium. The ice-nucleating temperature was -4.7°C for a non-treated control (sterilized distilled water), whereas it was -9.6°C for an INA- bacterium Escherichia coli TOP10. These methods detected pathogenic strains from apple orchards. Pss might exist in an apple tree during ice injury, and it secretes a toxin that makes leaves yellow and cause canker symptoms. Until now, Korea has not developed antibiotics targeting Pss. Therefore, it is necessary to develop effective disease control to combat Pss in apple orchards. Pathogenicity test on apple leaves and stems showed canker symptoms. The pathogenic bacterium was re-isolated from symptomatic plant tissue and confirmed as original isolates by 16S rRNA. Repetitive element sequence-based PCR and enterobacterial repetitive intergenic consensus PCR primers revealed different genetic profiles within P. syringae pathovars. High antibiotic susceptibility results showed the misreading of mRNA caused by streptomycin and oxytetracycline.

Genome-wide characterization of the chitinase gene family in wild apple (Malus sieversii) and domesticated apple (Malus domestica) reveals its role in resistance to Valsa mali.

Chitinases are responsible for catalyzing the hydrolysis of chitin and contribute to plant defense against fungal pathogens by degrading fungal chitin. In this study, genome-wide identification of the chitinase gene family of wild apple (Malus sieversii) and domesticated apple (Malus domestica) was conducted, and the expression profile was analyzed in response to Valsa mali infection. A total of 36 and 47 chitinase genes belonging to the glycosyl hydrolase 18 (GH18) and 19 (GH19) families were identified in the genomes of M. sieversii and M. domestica, respectively. These genes were classified into five classes based on their phylogenetic relationships and conserved catalytic domains. The genes were randomly distributed on the chromosomes and exhibited expansion by tandem and segmental duplication. Eight of the 36 MsChi genes and 17 of the 47 MdChi genes were differentially expressed in response to V. mali inoculation. In particular, MsChi35 and its ortholog MdChi41, a class IV chitinase, were constitutively expressed at high levels in M. sieversii and domesticated apple, respectively, and may play a crucial role in the defense response against V. mali. These results improve knowledge of the chitinase gene family in apple species and provide a foundation for further studies of fungal disease prevention in apple.

Apple endophyte community in relation to location, scion and rootstock genotypes and susceptibility to European canker.

European apple canker, caused by Neonectria ditissima, is a severe disease of apple. Achieving effective control is difficult with the currently available pesticides. Specific apple endophytes associated with cultivars may partially contribute to the cultivar response to the pathogen and thus could be used for disease management. We sought to determine whether the overall endophyte community differed among cultivars differing in their susceptibility to N. ditissima and to identify specific microbial groups associated with the susceptibility. Using Illumina MiSeq meta-barcoding, we profiled apple tree endophytes in 16 scion-rootstock combinations at two locations and quantified the relative contribution of scion, rootstock and location to the observed variability in the endophyte communities. Endophyte diversity was primarily affected by the orchard location (accounting for 29.4% and 85.9% of the total variation in the PC1 for bacteria and fungi, respectively), followed by the scion genotype (24.3% and 19.5% of PC2), whereas rootstock effects were small (<3% of PC1 and PC2). There were significant differences in the endophyte community between canker-resistant and -susceptible cultivars. Several bacterial and fungal endophyte groups had different relative abundance between susceptible and resistant cultivars. These endophyte groups included putative pathogen antagonists as well as plant pathogens. Their possible ecological roles in the N. ditissima pathosystem are discussed.

The Effects of Arbuscular Mycorrhizal Fungal Colonisation on Nutrient Status, Growth, Productivity, and Canker Resistance of Apple (Malus pumila).

We assess whether arbuscular mycorrhizal fungi (AMF) improve growth, nutritional status, phenology, flower and fruit production, and disease resistance in woody perennial crops using apple (Malus pumila) as a study system. In a fully factorial experiment, young trees were grown for 3 years with or without AMF (Funneliformis mosseae and Rhizophagus irregularis), and with industrial standard fertiliser applications or restricted fertiliser (10% of standard). We use two commercial scions (Dabinett and Michelin) and rootstocks (MM111 and MM106). Industrial standard fertiliser applications reduced AMF colonisation and root biomass, potentially increasing drought sensitivity. Mycorrhizal status was influenced by above ground genotypes (scion type) but not rootstocks, indicating strong interactions between above and below ground plant tissue. The AMF inoculation significantly increased resistance to Neonectria ditissima, a globally economically significant fungal pathogen of apple orchards, but did not consistently alter leaf nutrients, growth, phenology or fruit and flower production. This study significantly advances understanding of AMF benefits to woody perennial crops, especially increased disease resistance which we show is not due to improved tree nutrition or drought alleviation. Breeding programmes and standard management practises can limit the potential for these benefits.