Molecular Phylogeny of Cytospora Species Associated with Canker Diseases of Apple Trees in Türkiye

Apple (Malus domestica) is a very important crop grown in Kazakhstan. Cytospora species are capable of causing destructive stem cankers on a wide range of woody plants, including apples, and can lead to twig and branch dieback. This study aimed to identify the Cytospora species responsible for canker disease of apple in Kazakhstan and to assess the susceptibility of major apple cultivars to these pathogens. Investigations were conducted in Almaty, Kazakhstan, during 2023 and 2024. Samples from symptomatic trees were collected, and nine Cytospora isolates were obtained from diseased apple trees. Multigene phylogenetic analysis based on combined sequence data of ITS, tef1-α, tub2, and LSU loci, together with morphological characteristics and pathogenicity assays, revealed two Cytospora species: C. leucostoma and C. sorbicola. The reactions of six apple cultivars (Gala, Golden Delicious, Red Delicious, Granny Smith, Fuji, and Jonaprince) to these species were evaluated, and statistically significant differences were found among cultivars (p < 0.05). The largest lesions occurred on Red Delicious and Fuji, indicating that these cultivars are the most susceptible. In contrast, lesion lengths on Jonaprince were significantly smaller than on all other cultivars, suggesting that Jonaprince is resistant to Cytospora species in Kazakhstan. This is the first report of C. leucostoma and C. sorbicola causing apple canker disease in Kazakhstan.

Multigene phylogeny, morphology, and pathogenicity trials reveal novel Cytospora species involved in perennial canker disease of apple trees in Iran

Cytospora canker (CC) is among the most important diseases in conifer trees (Picea spp., mainly). This disease poses a significant risk factor for forest health, potentially leading to economic losses for wood producers. To provide a genomic basis of the CC pathogenesis, the genomes of two Cytospora species associated with the disease were sequenced and further analyzed within a set of Diaporthales species. The first species was identified as C.piceae. The second was described as C.piceicola sp. nov. based on morphological characteristics and multi-gene phylogenetic analysis. The novel species is sister to other Cytospora species isolated from conifers. Here, we report 39.7 and 43.8 Mb highly contiguous genome assemblies of C.piceae EI-19(A) and C.piceicola EI-20, respectively, obtained using Illumina sequencing technology. Despite notably different genome sizes, these species share the main genome characteristics, such as predicted gene number (10,862 and 10,742) and assembly completeness (97.6% and 98.1%). A wide range of genes encoding carbohydrate-active enzymes, secondary metabolite biosynthesis clusters, and secreted effectors were found. Multiple experimentally validated virulence genes were also identified in the studied species. The defined arsenals of enzymes and effectors generally relate to the hemibiotrophic lifestyle with a capability to switch to biotrophy. The obtained evidence also supports that C.piceae EI-19(A) and C.piceicola EI-20 can cause severe canker disease symptoms in Picea spp. specifically. It was additionally observed that the strains of C.piceae may have different pathogenicity and virulence characteristics based on the analyses of predicted secondary metabolite complements, effectomes, and virulence-related genes. Phylogenomic analysis and timetree estimations indicated that divergence of the studied species may have occurred relatively late, 11-10 million years ago. Compared to other members of Diaporthales, C.piceae EI-19(A) and C.piceicola EI-20 implied a moderate rate of gene contraction, but the latter experienced significant gene loss that can additionally support host specificity attributed to these species. But uncovered gene contraction events may point out potential lifestyle differentiation and host shift of the studied species. It was revealed that EI-19(A) and C.piceicola EI-20 carry distinct secretomes and effectomes among Diaporthales species. This feature can indicate a species lifestyle and pathogenicity potential. These findings highlight potential targets for identification and/or detection of pathogenic Cytospora in conifers. The introduced draft genome sequences of C.piceae and C.piceicola can be employed as tools to understand basic genetics and pathogenicity mechanisms of fungal species causing canker disease in woody plants. The identified pathogenicity and virulence-related genes would serve as potential candidates for host-induced gene silencing aimed at making plant hosts more resistant to pathogenic species. Furthermore, the comparative genomics component of the study will facilitate the functional analysis of the genes of unknown function in all fungal pathogens.

SummaryA stem canker disease of apple that differs from previously reported canker diseases has been recorded in Xinjiang Uygur Autonomous Region in China. Eight Cytospora strains obtained from diseased apple trees and inoculated onto healthy apple tree branches resulted in the development of the same disease symptoms. This pathogen formed conceptacles, which were multilocular pycnidia with common walls and a single ostiole. Comprehensive analysis of three DNA sequence regions (internal transcribed spacer, large ribosomal subunit and transcription elongation factor) and morphological and cultural characteristics revealed that these strains were most similar to the fungus Cytospora parasitica. This is the first report of C. parasitica causing canker disease of apple in China.

During the year 2015 and 2016, we surveyed the main pistachio growing counties in California to assess the health of pistachio trees and investigate the occurrence of canker and soil borne diseases. Numerous orchards were visited throughout the San Joaquin Valley to diagnose unusual symptoms and investigate the causes of decline of pistachio trees. Canker diseases causing branch and scaffold dieback were observed commonly in mature pistachio orchards. New fungal pathogens associated with cankers included Cytospora species, Diaporthe ambigua (=Phomopsis ambigua), and Colletotrichum karstii. A pathogenicity test using fungi isolated from cankers was conducted in the field to determine the ability of these fungi to produce cankers. Results of this study showed that all species tested were pathogenic to pistachio, causing extensive lesions and vascular discolorations just a few months after infection. Among them, Cytospora species were the most aggressive fungal pathogens and are considered new threats to pistachio in California. Other disease symptoms observed commonly in our surveys included declining trees characterized by chlorotic foliage as well as wilting, defoliation and eventual tree death. Trunks often expressed gumming together with crown rot symptoms. Investigations of the causes of pistachio tree decline revealed the occurrence in orchards of several Phytophthora species including P. niederhauserii, P. cinnamomi and P. taxon walnut. Other fungi isolated from declining pistachio trees and rootstocks included Fusarium oxysporum, F. solani, F. equiseti and F. proliferatum and Macrophomina phaseolina. Pathogenicity in pistachio of the various soil borne pathogens were conducted in potted UCB-1 plants maintained in a greenhouse. Pathogenicity studies showed that all the various fungi can colonize and damage the vascular tissues of UCB-1 seedling rootstocks, causing substantial lesions and vascular discolorations. To date, the present project allowed to identify several new pathogens currently affecting the health of pistachio trees in California.

The occurrence of apple replant disease (ARD) is closely related to the increase of soil pathogenic fungi abundance. However, the relationship between ARD and fungal community structure remains poorly understood. In this study, Illumina high-throughput sequencing was used to investigate the composition, diversity, and function of rhizosphere fungal communities associated with healthy (HRS) and diseased apple trees (DRS). Microbial taxa related to ARD were also identified. The severity of ARD varied among the sampled orchards. We found that Ascomycota was the dominant phylum in the DRS fungal taxa, and the fungal community abundance and Simpson index of DRS were significantly higher than those of HRS. Cluster and FUNGuild database analyses revealed significant differences in the relative abundance and function of fungal taxa between DRS and HRS. Most fungi isolated from DRS were plant pathogens, predominantly from the genus Fusarium (Ascomycota, Nectriaceae), which was also the predominant fungal genus detected in DRS. In contrast, Mortierella was more abundant in HRS. To validate the sequencing results, Fusarium isolates, including F. proliferatum, F. oxysporum, and F. solani, were verified as pathogens and showed high virulence. Structural equation modeling indicated that the occurrence of ARD was directly or indirectly influenced by Fusarium, Mortierella, phloridin, available phosphorus, and soil organic matter. Further research is needed to elucidate how soil parameters affect ARD. Laboratory tests demonstrated that F. proliferatum MR5 can produce pectinase and cellulase and is sensitive to two fungicides: flusilazole and bromothalonil. In conclusion, the deterioration of rhizosphere fungal community structure may be a key biological factor driving ARD, with Fusarium in DRS identified as a major causative agent of ARD in China. The findings of this study provide valuable insights for developing preventive strategies against ARD.Supplementary informationThe online version contains supplementary material available at 10.1007/s44154-025-00258-1.

Studies of canker and dieback of oak tree in China, with two <i>Cytospora</i> species described

Apples are widely consumed by people all over the world due to their taste and nutritional value. However, apple trees are prone to various environmental stresses, including fungal diseases. Among them, Cytospora canker (or Valsa canker) can cause dieback of branches and twigs. Although Kazakhstan is well known as an origin of apples, very little is known about canker diseases that spread across all commercial orchards. Therefore, an accurate identification of the causal agents of those diseases is needed for further application of informed disease management strategies. In this study, eleven isolates belonging to four Cytospora species were isolated from multiple cultivars, grown in six different orchards within the Almaty region, Kazakhstan. As a result of a multilocus phylogenetic analysis using ITS, LSU and tef1-α marker genes and morphological characterization, these isolates were described as Cytospora parasitica, Cytospora sorbina, Cytospora pruinopsis and Cytospora chrysosperma. Moreover, a pathogenicity test was conducted on detached twigs, and it demonstrated that two of these fungi were highly virulent. Overall, this paper is a first report of the causal agents of apple canker disease in Kazakhstan and could be a trigger for conducting future studies to better understand the disease epidemiology, as well as build management strategies.

Cytospora species associated with walnut canker disease in China, with description of a new species C. gigalocus

Cytospora species are widely distributed and often occur as endophytes, saprobes or phytopathogens. They primarily cause canker and dieback diseases of woody host plants, leading to the growth weakness or death of host plants, thereby causing significant economic and ecological losses. In order to reveal the diversity of Cytospora species associated with canker and dieback diseases of coniferous trees in China, we assessed 11 Cytospora spp. represented by 28 fungal strains from symptomatic branches or twigs of coniferous trees, i.e., Juniperus procumbens, J. przewalskii, Picea crassifolia, Pinus armandii, P. bungeana, Platycladus orientalis in China. Through morphological observations and multilocus phylogeny of ITS, LSU, act, rpb2, tef1-α, and tub2 gene sequences, we focused on four novel Cytospora species (C. albodisca, C. discostoma, C. donglingensis, and C. verrucosa) associated with Platycladus orientalis. This study represented the first attempt to clarify the taxonomy of Cytospora species associated with canker and dieback symptoms of coniferous trees in China.

Cytospora species are important phytopathogens causing severe canker disease with a worldwide distribution and broad host range. However, identification of taxa to species level is difficult due to poor phylogenetic understanding and lack of sequenced type species. Morphological and phylogenetic studies have been carried out on several important hosts such as Eucalyptus and Malus in China, Iran, and South Africa. In this study destructive canker diseases of the anti-desertification plants, Elaeagnus angustifolia, Hippophae rhamnoides, and Salix psammophila, were investigated in northwest China. Multilocus phylogenetic analyses of ITS, nrLSU, RPB2, and ACT gene regions, combined with detailed morphological analyses and comparison with ex-type strains revealed six Cytospora species, C. chrysosperma, C. elaeagni, C. hippophaes, C. nivea, C. populina comb. nov. and C. gigaspora sp. nov. causing cankers on these hosts. The novel species C. gigaspora has flat multiple locules with a conceptacle and unusually long 12 µm conidia. Detailed descriptions and molecular data for the Cytospora species causing cankers on the three psammophilic host plants are provided. Cytospora elaeagni and C. hippophaes have previously been recorded from Elaeagnus angustifolia and Hippophae rhamnoides, whereas the other species causing Cytospora canker of Elaeagnus angustifolia and Salix psammophila are new records.

Almond canker diseases are destructive and can reduce the yield as well as the lifespan of almond orchards. These diseases may affect the trunk and branches of both young and mature trees and can result in tree death soon after orchard establishment in severe cases. Between 2015 and 2018, 70 almond orchards were visited throughout the Central Valley of California upon requests from farm advisors for canker disease diagnosis. Two major canker diseases were identified, including Botryosphaeriaceae cankers and Ceratocystis canker. In addition, five less prevalent canker diseases were identified, including Cytospora, Eutypa, Diaporthe, Collophorina, and Pallidophorina canker. Seventy-four fungal isolates were selected for multilocus phylogenetic analyses of internal transcribed spacer region ITS1-5.8S-ITS2 and part of the translation elongation factor 1-α, β-tubulin, and glyceraldehyde 3-phosphate dehydrogenase gene sequences; 27 species were identified, including 12 Botryosphaeriaceae species, Ceratocystis destructans, five Cytospora species, Collophorina hispanica, four Diaporthe species, two Diatrype species, Eutypa lata, and Pallidophorina paarla. The most frequently isolated species were Ceratocystis destructans, Neoscytalidium dimidiatum, and Cytospora californica. Pathogenicity experiments on almond cultivar Nonpareil revealed that Neofusicoccum parvum, Neofusicoccum arbuti, and Neofusicoccum mediterraneum were the most virulent. Botryosphaeriaceae cankers were predominantly found in young orchards and symptoms were most prevalent on the trunks of trees. Ceratocystis canker was most commonly found in mature orchards and associated with symptoms found on trunks or large scaffold branches. This study provides a thorough examination of the diversity and pathogenicity of fungal pathogens associated with branch and trunk cankers of almond in California.

Apple scab (Venturia inaequalis (Cke) Wint) is the main disease of apple trees not only in the world but also in Turkey. The disease can cause severe yield losses in all Turkish apple orchards. Genetic diversity and relatedness of apple scab (Venturia inaequalis (Cke) Wint) isolates collected from Turkey and Europe were investigated in this study through molecular markers. RAPD, ISSR, SSR and SRAP markers were used in the molecular studies. Results obtained by marker system revealed that isolates were quite separated from each other and it is appeared to exist a variation between them. Genetic relatedness between the isolates are very close to each other, and difference among the groups is not significant due to host specificity and geographical location. The groups are consisted of more number of isolates when SSR and SRAP markers are used compared with ISSR and RAPD markers. Using SSR and SRAP markers are preferable to provide more informative outcomes because of ease of use, repeatability and specificity.

The causal agent of apple mosaic disease has been previously thought to be solely caused by apple mosaic virus (ApMV). In this study, we report that a novel ilarvirus is also associated with apple mosaic disease. Next-generation sequencing analysis of an apple tree showing mosaic symptoms revealed that the tree was infected with three apple latent viruses (apple stem pitting virus, apple stem grooving virus, and apple chlorotic leaf spot virus) and a novel ilarvirus (given the name apple necrotic mosaic virus (ApNMV)) that is closely related to Prunus necrotic ringspot virus (PNRSV) and ApMV. The genome of ApNMV consists of RNA1 (3378 nt), RNA2 (2767 nt), and RNA3 (1956 nt). A phylogenetic analysis based on the coat protein amino acid sequences indicated that the novel virus belongs to the same subgroup 3 of the genus Ilarvirus as PNRSV and ApMV. The presence of mosaic leaves, which tend to be unevenly distributed in diseased apple trees, was correlated with the internal distribution of ApNMV. RT-PCR detection of mosaic-diseased apple trees in Japan indicated that ApNMV was detected in apple trees introduced from China, whereas ApMV was detected from cultivated apple trees in domestic orchards. Consistent with these findings, a survey of mosaic-diseased apple trees in major apple-producing provinces in China revealed that the majority of apple trees showing mosaic symptoms in China are infected with ApNMV.

Apple tree disease is a main threat factor to apple quality and yield. This paper proposed an improved convolutional neural network model to classify apple tree diseases. It took the advantages of neural network to extract the deep characteristics of disease parts, and used deep learning to classify target disease areas. In order to improve the classification accuracy and speed up the convergence of the network model, the center loss and focal loss functions were fused, instead of the traditional softmax loss function, which was especially important for our classification network model. Experimental results on our apple trunk dataset showed that our model achieved an accuracy of 94.5%. Therefore our method is feasible and effective for apple tree disease identification.