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

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.

Oak (Quercus sp.) is an excellent tree species as a windbreak, for water conservation, and for fireproofing in forests in China. However, several trees of this genus were found to be suffering from various fungal diseases. In this study, we evaluated 15 fungal pathogens that can cause dieback and canker disease in oak in China, and discovered two Cytospora species. They were identified as Cytospora quercinum sp. nov. and C. vinacea, based on detailed morphological comparisons and phylogenetic analysis of ITS, LSU, act, rpb2, tef1‐α, and tub2 loci. This study is the first record of C. quercinum and C. vinacea as causal agents of dieback in oak based on pathogenicity tests conducted on 2‐year‐old plants in a greenhouse. In addition, this study also revealed the influence of different conditions on the growth rate of mycelia. Mycelial growth of C. quercinum and C. vinacea occurred at optimum temperatures of 20.1 and 20.8 °C, and optimum pH of 5.4 and 5.3, respectively. For these two species, utilization of glucose and fructose was highly efficient, and sucrose was the least efficient. The habitat of Quercus mongolica indicated that more attention and management are needed to prevent the occurrence of Cytospora disease in the summer in north‐eastern China, and in spring and autumn in eastern and northern China. This study contributes to the understanding of the species causing canker or dieback diseases in important economic forest trees, and provides useful information for effective disease management of oak trees in China.

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.

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.

Canker and wood rot pathogens cause dieback and, in severe cases, the death of young apple trees. Recently, a higher occurrence of cankers was observed on 1-year-old apple trees in the Western Cape Province of South Africa. This study aimed to assess the phytosanitary status of nursery trees and propagation material as possible inoculum sources for canker pathogens. Thirteen 1-year-old apple orchards showing canker or dieback symptoms were sampled. Certified nursery apple trees were collected from four nurseries as well as scion and rootstock mother plant material. Isolations were made from the discoloration observed in the vascular tissue of the plant parts and from asymptomatic material. Possible canker and wood rot species were identified with PCR and sequence comparisons of the relevant gene regions and phylogenetic analyses. Similar canker and wood rot species were isolated from 1-year-old diseased apple trees, nursery apple trees, and the propagation material. Forty-five fungal species associated with canker or wood rot symptoms were identified. The top five most abundant fungal species found causing disease on commercial 1-year-old trees were also found in high numbers causing latent infection in certified apple nursery trees. These species were Didymosphaeria rubi-ulmifolii sensu lato, Diplodia seriata, Schizophyllum commune, Didymella pomorum, and Coniochaeta fasciculata, with D. rubi-ulmifolii sensu lato being the dominant species in both sampling materials. In all, 65% of certified nursery apple trees, 5% of scion shoots used for budding, and 21% of rooted rootstock cuttings from layer blocks had latent infections of canker and wood rot pathogens. Pathogenicity trials were conducted with isolates of 39 species, inoculated onto 2-year-old branches of 14-year-old Golden Delicious trees. All species caused lesions that were significantly longer than the control. This study confirmed the presence of canker and wood rot pathogens in apple propagation material as well as certified nursery apple trees, which will aid the improvement of management practices in nurseries.

Background‘Rapid Apple Decline’ (RAD) is a newly emerging problem of young, dwarf apple trees in the Northeastern USA. The affected trees show trunk necrosis, cracking and canker before collapse in summer. In this study, we discovered and characterized a new luteovirus from apple trees in RAD-affected orchards using high-throughput sequencing (HTS) technology and subsequent Sanger sequencing.MethodsIllumina NextSeq sequencing was applied to total RNAs prepared from three diseased apple trees. Sequence reads were de novo assembled, and contigs were annotated by BLASTx. RT-PCR and 5′/3’ RACE sequencing were used to obtain the complete genome of a new virus. RT-PCR was used to detect the virus.ResultsThree common apple viruses and a new luteovirus were identified from the diseased trees by HTS and RT-PCR. Sequence analyses of the complete genome of the new virus show that it is a new species of the genus Luteovirus in the family Luteoviridae. The virus is graft transmissible and detected by RT-PCR in apple trees in a couple of orchards.ConclusionsA new luteovirus and/or three known viruses were found to be associated with RAD. Molecular characterization of the new luteovirus provides important information for further investigation of its distribution and etiological role.

Background/Objectives: Branch dieback and canker diseases caused by Cytospora species adversely impact the health of woody plants worldwide. Results: During this survey, 59 Cytospora isolates were obtained from symptomatic trees and shrubs growing in southwest Ontario and Saskatchewan, Canada. A DNA barcoding approach combined with morphological characterization identified 15 known species of Cytospora associated with these diseases: C. chrysosperma, C. curvata, C. euonymina, C. hoffmannii, C. kantschavelii, C. leucosperma, C. leucostoma, C. nitschkeana, C. piceae, C. populina, C. pruinopsis, C. pruinosa, C. ribis, C. schulzeri, and C. sorbina. The most common species isolated from multiple hosts were C. sorbina (10), C. chrysosperma (8), C. nitschkeana (6), and C. pruinosa (6). A wide range of host associations, including non-conifer species, was observed for C. piceae. Conclusions: The obtained results contribute to the study of diversity, host affiliation, geographical distribution, and pathogenicity of Cytospora species occurring on woody plants in both natural habitats and agricultural systems. The findings support the effectiveness of using DNA barcodes in fungal taxonomy and plant pathology studies.