Diversity and aggressiveness of the Diaporthe species complex on sunflower in Serbia.

This study aimed to investigate the Diaporthe species associated with Phomopsis stem canker of sunflower (Helianthus annuus L.) in Serbia. The significant increase in sunflower and soybean (Glycine max (L.) Merr.) cultivation may have created the bridge favorable conditions for the distribution of Diaporthe species in this region. The present study identified five Diaporthe species on sunflower: D. gulyae, D. helianthi, D. pseudolongicolla, D. stewartii, and the newly identified D. riccionae based on morphological, molecular, and pathogenic characteristics. The research emphasizes the importance of effective inoculation methods and evaluates the aggressiveness of isolates. Sunflower plants were inoculated using the stem wound method, while seeds of sunflower and soybean were inoculated using the standard seed method. Most of the tested isolates demonstrated high aggressiveness, resulting in over 80% premature wilting of sunflower plants. Additionally, this research examined the aggressiveness of Diaporthe species on sunflower seeds, highlighting D. stewartii and D. pseudolongicolla as common pathogens of both sunflower and soybean. The most aggressive species on seeds was D. stewartii, causing seed decay of up to 100% in sunflower and 97% in soybean. The findings suggest the development of resilient sunflower genotypes through breeding programs and the implementation of strategies to manage cross-contamination risks between sunflower and soybean crops. Furthermore, this study provides insights into the interactions between Diaporthe species and the seeds of sunflower and soybean. Future research will enhance our understanding of the impact of Diaporthe species on sunflower and soybean.

First Report of Stem Canker Disease Caused by Neoscytalidium dimidiatum on Euphrates Poplar in Xinjiang, China.

Euphrates poplar (Populus euphratica Oliv.) constitutes about 61% of the global poplar population, thriving in arid regions of western China (Wu et al. 2023). It plays a crucial role in maintaining ecological balance, securing oasis agriculture, and driving socio-economic progress in the region. During a June 2023 investigation in the P. euphratica forest within the Hotan area of Xinjiang (37°20'21″N, 79°21'15″E), over 12% of the P. euphratica trees displayed branch withering symptoms. The affected trees exhibited cracked bark, trunk decay, darkened coloration, and an eventual black coal-smoke-like appearance. Fungal spores were notably present beneath peeling bark on trunks and main branches. The deep ulcers extended longitudinally into the cambium, leading to tree mortality. In some cases, lateral spread into the sapwood caused dark discoloration of vascular tissue. Twenty diseased branches from various locations were collected and 5-10 mm2 lesions were excised from the edges. These were then surface-disinfected with 75% ethanol for 30 s and 1% sodium hypochlorite for 2 min. After three rinses with sterile distilled water, excess moisture was removed using sterile filter paper, followed by incubating the samples on Potato Dextrose Agar (PDA) medium. Cultures were subsequently grown at 25 ± 1 ℃ under a 12-h photoperiod for three days, thus resulting in the isolation of 25 fungal strains with similar morphological characteristics. All strains displayed rapid colony growth (40 mm/d). On PDA medium, the mycelium initially presented as a white colony, transitioning to an olive-green to greyish color, finally turning dark-grey to black. Colonies generated mycelia that disintegrated into 0- to 1-septate, cylindrical to round, hyaline to brown arthroconidia, occurring singly or in arthric chains, averaging 8.9 ± 2.1 µm × 4.9 ± 1.3 µm, with a length/width ratio of 1.79. Based on morphological characteristics, the isolates were identified as Neoscytalidium dimidiatum (Penz.) Crous & Slippers (Crous et al. 2006). Molecular characterization involved amplifying the partial internal transcribed spacer (ITS) region and translation elongation factor 1-α (TEF1-α) and ß-tubulin (TUB2) genes using ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and BT2a/BT2b primers (Glass and Donaldson 1995). Sequences, available in GenBank (ITS: PP033096, PP033097, PP033098; TUB2: PP032812, PP032813, PP032814; TEF1-α: PP032815, PP032816, PP032817), exhibited 99-100% identity with the epitype N. dimidiatum Arp2-D (ITS, MK813852; TUB2, MK816354; TEF1-α, MK816355). Phylogenetic analysis, employing maximum likelihood and Bayesian inference on concatenated ITS-TEF1-TUB, was constructed using IQ-Tree and MrBayes3.2.7, revealing isolates clustering within the N. dimidiatum clade. Three isolates (HY01, HY02, and HY05) from different collection points were chosen for pathogenic investigation. Pathogenicity testing on one-year-old healthy P. euphratica seedlings involved removing a 4-mm-diameter bark plug using a cork borer. A 3-day-cultured N. dimidiatum plug of the same size was inoculated, with a blank PDA as control. The wound was covered with moistened sterile absorbent cotton and finally sealed with parafilm for three days. Experiments were repeated thrice. Symptoms manifested by day 2 post-inoculation, resembling the original symptoms by day 7. In the control group, plants remained healthy. N. dimidiatum was exclusively re-isolated from lesions on inoculated stems, confirmed as N. dimidiatum through morphological characteristics and sequence analysis, aligning with Koch's hypothesis. To our knowledge, this is the first report of N. dimidiatum inducing stem canker on P. euphratica in China. This pathogen has been reported on many tree hosts including citrus (Alananbeh et al., 2020), common fig (Güney et al., 2022), dragon fruit (Salunkhe et al., 2023), and Almond (Nouri et al., 2018). Therefore our findings will serve as a warning for authorities to a potential threat in China's P. euphratica and other trees cultivation. Thus, further epidemiological studies are essential for devising effective management strategies.

Effect of Fungicide and Timing of Application on Management of Phoma Black Stem of Cultivated Sunflowers in the United States.

Phoma black stem (PBS), caused by Phoma macdonaldii Boerema (teleomorph Leptosphaeria lindquistii Frezzi), is the most common stem disease of sunflower (Helianthus annuus L.) in the northern Great Plains region of the United States. However, the impact of PBS on sunflower yield in the United States is unclear, and a near complete absence of information on the impact of fungicides on disease management exists. The objectives of this study were to determine the impact of PBS on sunflower yield, the efficacy of available fungicides, the optimal fungicide application timing, and the economic viability of fungicides as a management tool. Fungicide timing efficacy was evaluated by applying single and/or sequential applications of pyraclostrobin fungicide at three sunflower growth stages in 10 field trials between 2017 and 2019. Efficacy of 10 fungicides from the Fungicide Resistance Action Committee (FRAC) groups 3, 7, and 11 were evaluated in four field trials between 2018 and 2019. The impact of treatments on PBS were evaluated by determination of incidence, severity, maximum lesion height, disease severity index (DSI), and harvested yield. Nine of the 10 fungicides evaluated and all fungicide timings that included an early bud application resulted in disease reductions when compared with the nontreated controls. The DSI was negatively correlated to sunflower yield in high-yield environments (P = 0.0004; R2 = 0.3425) but not in low- or moderate-yield environments. Although FRAC 7 fungicides were generally most efficacious, the sufficient efficacy and lower cost of FRAC 11 fungicides make them more economically viable in high-yielding environments at current market conditions.

Deep amplicon sequencing reveals extensive allelic diversity in the erg11/CYP51 promoter and allows multi-population DMI fungicide resistance monitoring in the canola pathogen Leptosphaeria maculans.

Continued use of fungicides provides a strong selection pressure towards strains with mutations to render these chemicals less effective. Previous research has shown that resistance to the demethylation inhibitor (DMI) fungicides, which target ergosterol synthesis, in the canola pathogen Leptosphaeria maculans has emerged in Australia and Europe. The change in fungicide sensitivity of individual isolates was found to be due to DNA insertions into the promoter of the erg11/CYP51 DMI target gene. Whether or not these were the only types of mutations and how prevalent they were in Australian populations was explored in the current study. New isolates with reduced DMI sensitivity were obtained from screens on DMI-treated plants, revealing eight independent insertions in the erg11 promoter. A novel deep amplicon sequencing approach applied to populations of ascospores fired from stubble identified an additional undetected insertion allele and quantified the frequencies of all known insertions, suggesting that, at least in the samples processed, the combined frequency of resistant alleles is between 0.0376% and 32.6%. Combined insertion allele frequencies positively correlated with population-level measures of in planta resistance to four different DMI treatments. Additionally, there was no evidence for erg11 coding mutations playing a role in conferring resistance in Australian populations. This research provides a key method for assessing fungicide resistance frequency in stubble-borne populations of plant pathogens and a baseline from which additional surveillance can be conducted in L. maculans. Whether or not the observed resistance allele frequencies are associated with loss of effective disease control in the field remains to be established.

The genome of a Far Eastern isolate of Diaporthe caulivora, a soybean fungal pathogen.

Diaporthe caulivora is an economically important fungal pathogen and a causal agent of soybean stem canker and seed decay. Here, the genome of a Russian Far Eastern isolate of D. caulivora was sequenced, assembled, and announced. Assembly quality was enough for advanced annotation, including prediction of potential disease-related genes encoding virulence factors and molecular determinants contributing to pathogen-host selection, interactions, and adaptation. Comparative analysis of 15 Diaporthe species was conducted regarding general genome properties, collinearity, and proteomes, and included detailed investigation of interspersed repeats. A notable feature of this analysis is a high recombinant variability of Diaporthe genomes, determined by the number and distribution of interspersed repeats, which also proved to be responsible for the diversity of GC content and genome size. This variability is assumed the main determinant of the divergence of Diaporthe genomes. A Bayesian multi-gene phylogeny was inferred for the 15 Diaporthe species on the basis of twenty thousand polymorphic sites of > 100 orthologous genes using independently adjusted evolutionary models. This allowed for the most accurate determination of evolutionary relationships and species boundaries for effective reporting about these plant pathogens. The evidence, obtained by different genome analysis techniques, implies the host-independent evolution of Diaporthe species. KEY POINTS: • The genome of a Far Eastern isolate of D. caulivora was announced. • A high degree of recombinant variability determines genomic divergence in Diaporthe genus. • The multi-gene phylogeny implies host-independent evolution of Diaporthe species.

Polymorphism of Avirulence Genes and Adaptation to Brassica Resistance Genes Is Gene-Dependent in the Phytopathogenic Fungus Leptosphaeria maculans.

The fungal phytopathogen Leptosphaeria maculans, which causes stem canker (blackleg) of rapeseed (Brassica napus), is mainly controlled worldwide by genetic resistance, which includes major resistance genes (Rlm). This model is one of those for which the highest number of avirulence genes (AvrLm) has been cloned. In many systems, including the L. maculans-B. napus interaction, intense use of resistance genes exerts strong selection pressure on the corresponding avirulent isolates, and the fungi may rapidly escape resistance through various molecular events which modify the avirulence genes. In the literature, the study of polymorphism at avirulence loci is often focused on single genes under selection pressure. In this study, we investigate allelic polymorphism at 11 avirulence loci in a French population of 89 L. maculans isolates collected on a trap cultivar in four geographic locations in the 2017-2018 cropping season. The corresponding Rlm genes have been (i) used for a long time, (ii) recently used, or (iii) unused in agricultural practice. The sequence data generated indicate an extreme diversity of situations. For example, genes submitted to an ancient selection may have either been deleted in populations (AvrLm1) or replaced by a single-nucleotide mutated virulent version (AvrLm2, AvrLm5-9). Genes that have never been under selection may either be nearly invariant (AvrLm6, AvrLm10A, AvrLm10B), exhibit rare deletions (AvrLm11, AvrLm14), or display a high diversity of alleles and isoforms (AvrLmS-Lep2). These data suggest that the evolutionary trajectory of avirulence/virulence alleles is gene-dependent and independent of selection pressure in L. maculans. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Foliar Fungicides Containing FRAC 11 Mitigate Phomopsis Stem Canker in Sunflower (Helianthus annuus).

Phomopsis stem canker reduces yield of sunflower (Helianthus annuus L.) up to or exceeding 40%; however, management recommendations have not been developed for U.S. farmers. Between 2009 and 2020, foliar fungicide trials were conducted in Minnesota, Nebraska, North Dakota, and South Dakota for a total of 49 location-years. Random effects meta-analyses were performed on the disease severity index (DSI) and yield data collected from the foliar fungicide trials to determine the overall and individual effectiveness of the tested fungicides. Effect sizes, Cohen's f or Hedges' g, were calculated as the difference in DSI or yield between the fungicide treatment and nontreated control (NTC) divided by the pooled SD. The pooled Cohen's f for DSI and yield was 0.40 (95% CI = [0.29, 0.42]), indicating a large effect size and that fungicide treatments had a significant effect on DSI and yield (P < 0.0001). Among the fungicide groups, quinone outside inhibitor (QoI) (DSI [k = 45; g = -0.47] and yield [k = 46; g = 0.41]) is moderately effective and premixes of demethylation inhibitors (DMI), succinate dehydrogenase inhibitors (SDHI), and QoI (DMI + SDHI + QoI) (DSI [k = 3; g = -0.79] and yield [k = 3; g = 0.94]) are largely effective in comparison with NTC. Upon performing prediction analyses, the probability of not recovering the fungicide application cost (Ploss) associated with QoI (pyraclostrobin) was <0.35 for a range of sunflower grain prices suggesting a greater probability of return on investment from a single application of fungicide. Overall, our study suggests that the use of QoI fungicides is likely to be profitable in the presence of Phomopsis stem canker (DSI > 5%).

First report of Diaporthe sojae causing stem canker on blueberry in China.

Blueberry (Vaccinium corymbosum) plants are popular all over the world due to their high nutritional value and health benefits. In October 2020, blueberry stems (cv. O'Neal) displaying reddish brown necrotic lesions were observed from a blueberry field in Anqing (Anhui, China), with the incidence of approximately 90%. The affected plants were somewhat stunted that had smaller fruit, and in severe cases, partial or whole plant died. We randomly selected three sampling sites to collect stems with the symptoms. Samples at the margin between diseased and healthy tissues were taken out, cut into 5 mm pieces in length，and then mixed them together. Twenty small samples were surface-sterilized, and plated onto potato dextrose agar (PDA). The plates were incubated at 25°C in the dark until fungal colonies were observed. After subculturing single hyphal tips, 9 out of 12 fungal isolates with similar morphologies were obtained. The representative isolate, LMKY12 was selected for further identification. The colonies on PDA showed white, fluffy aerial mycelia with 7.9  0.2 mm (n=5) diameter after inoculation in darkness at 25°C for one week. The colony darkens in color with age, yellowish pigmentation in reverse were observed. After 15 days of incubation, dark brown, irregular hard particles (fruiting bodies in sexual stage) accumulated on the surface of the colonies. Asci were 8-spored, sessile, club-like, hyaline, and 35-46 x 6-9 µm (n=30) in size. The ascospores were oval or spindle shaped, two-celled, constricted at division, and containing four guttulates with larger guttules at centre and smaller one at ends, measured 9-11 x 2-4 um (n=50). No sporulation observed on blueberry stems after inoculated 30 days. In order to induce the production of conidiophores, mycelial plugs were placed on blueberry leaves and cultured in darkness at 25°C. There are two types of conidia observed after 20 days of inoculation. Alpha conidia were aseptate, hyaline, smooth, ovate to ellipsoidal, often biguttulate, measured 5.33-7.26 x 1.65-2.53 µm (n=50). Beta conidia were hyaline, linear, measured 12.60-17.91 x 0.81-1.38 µm (n=30). The morphological characteristics matched the previous description of D. sojae (Udayanga et al. 2015; Guo et al. 2020). To confirm the identification, the mycelial genomic DNA of LMKY12 was extracted as a template. The rDNA internal transcribed spacer (ITS), translation elongation factor 1-α gene (TEF1-α), and calmodulin (CAL) were amplified and sequenced using primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R, and CAL-228F/CAL-737R (Carbone and Kohn 1999), respectively. BLAST analysis revealed that the ITS (ON545758), CAL (OP886852), and TEF1-α (OP886853) sequences were 100% (527/527 base pairs), 99.21% (504/508 base pairs), and 99.41% (336/338 base pairs) similar to the strain FAU636 of D. sojae (KJ590718, KJ612115, KJ590761), respectively. Phylogenetic analysis based on concatenated sequences of ITS, TEF1-α, and CAL using MEGA 7.0 by maximum likelihood attributed the isolate LMKY12 to the D. sojae clade. Pathogenicity tests were performed on blueberry cv. O'Neal using detached stems (n=8) in laboratory, one-year-old potted plants (n=4) in greenhouse. Inoculations were done by placing mycelial plugs (7 mm in diameter) taken from a 7-day-old PDA culture on wounded stems. Inoculations with uncolonized agar plugs served as negative controls. Reddish dark brown lesions similar to the symptoms were observed on all inoculated stems 7 days after inoculation. No symptoms developed on control stems. Reisolations were successfully made from all the inoculated stems, and the pathogen was confirmed by the presence of pycnidia, alpha conidia and beta conidia. To our knowledge, this is the first report of D. sojae causing blueberry stem canker in China.

Pestalotiopsis Species Associated with Blueberry Leaf Spots and Stem Cankers in Sichuan Province of China.

Blueberry leaf spots and stem cankers caused by Pestalotiopsis spp. have become a serious threat for the production of blueberry in Sichuan Province. To characterize the etiology of the diseases connected with these fungi, samples showing leaf spot and stem canker symptoms were collected from the 12 main blueberry-growing areas of Sichuan Province from 2015 to 2020 and used for pathogen isolation. In total, 91 fungal isolates were obtained with preliminary morphological identification and 48 representative strains were selected for further pathogenicity test and molecular identification. Four species, including Pestalotiopsis clavispora (Neopestalotiopsis clavispora) (57.14%), P. trachicarpicola (28.57%), P. chamaeropis (13.19%), and P. adusta (1.10%), were identified based on conidial morphology, cultural characteristics, and phylogenetic analysis of the internal transcribed spacer region, partial sequence of the ß-tubulin gene, and the translation elongation factor 1-α. Pathogenicity tests showed that four species were pathogenic to leaves and stems of blueberry. Among them, P. clavispora (N. clavispora) was the most aggressive as the predominant species to cause both leaf spot and stem canker. P. trachicarpicola and P. chamaeropis were mainly isolated from leaves but also pathogenic to stems. P. adusta was only isolated from stems but also pathogenic to leaves. To the best of our knowledge, this is the first report of P. chamaeropis and P. adusta as pathogens causing leaf spots and stem canker on blueberry. The results provide helpful information in disease diagnosis and management of blueberry.

Multifaceted plant growth-promoting traits of indigenous rhizospheric microbes against Phomopsis theae, a causal agent of stem canker in tea plants.

Phomopsis canker is one of the major devastating stem diseases that occur in tea plants caused by the fungal pathogen Phomopsis theae. Rapid development of this disease leads to a capital loss in the tea industry which demands an ecofriendly disease management strategy to control this aggressive pathogen. A total of 245 isolates were recovered from the tea rhizosphere and screened for in vitro plant growth promoting (PGP) traits and antagonism against P. theae. Among them, twelve isolates exhibited multifarious PGP traits including phytohormones, siderophore, hydrogen cyanide, salicylic acid production, phosphate solubilization, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, and antifungal activity. In vitro studies on morphological, biochemical, and phylogenetic analyses classified the selected isolates as Pseudomonas fluorescens (VPF5), Bacillus subtilis (VBS3), Streptomyces griseus (VSG4) and Trichoderma viride (VTV7). Specifically, P. fluorescens VPF5 and B. subtilis VBS3 strains showed the highest level of PGP activities. On the other hand, VBS3 and VTV7 strains showed higher biocontrol efficacy in inhibiting mycelia growth and spore germination of P. theae. A detailed investigation on hydrolytic enzymes produced by antagonistic strains, which degrade the fungus cell wall, revealed that highest amount of chitinase and ß-1,3- glucanase in VTV7 and VBS3 strains. Further, the key antifungal secondary metabolites from these biocontrol agents associated with suppression of P. theae were identified using gas chromatography mass spectrometry. The above study clearly recognized the specific traits in the isolated microbes, which make them good candidates as plant growth-promoting rhizobacteria (PGPR) and biocontrol agents to improve plant growth and health. However, greenhouse trials and field application of these beneficial microbes is required to further confirm their efficacy for the management of stem canker in tea cultivation.

First Report of Stem Canker on Dangshen (Codonopsis pilosula) Caused by Binucleate Rhizoctonia AG-K in China.

Dangshen (Codonopsis pilosula) is a well-known medicinal and food homologous plant in China, which is widely used as a tonic agent and has good immunomodulatory effects (Bai et al. 2020; Luan et al. 2021). To retain the best medicinal properties, growers imitated the original ecological planting method for cultivating C. pilosula in hillside fields in Wutai county, Shanxi province, China. In July and August 2021 and 2022, stem canker disease was observed in C. pilosula. The basal part of the stems showed slightly sunken brown lesions, and the disease incidence was up to 20% in the investigated fields (6.67 ha). To identify the causal agents of stem canker, 12 small pieces (approximately 5 mm long) from 12 diseased samples (one piece per sample) were cut from the border of the lesions, surface-sterilized (70% ethanol for 30 s, 0.5% NaClO for 3 min), washed three times with sterile water, and then incubated on water agar (WA) at 25 °C for 24 h. Isolates with right-angle branching, a septum near the branch, and a slight constriction at the branch base were selected, and their hyphal tips were transferred onto potato dextrose agar (PDA) plates. After incubation at 25 °C, 12 Rhizoctonia-like isolates (Dcp-19 to Dcp-30) with white colonies were obtained. White monilioid cells in aerial mycelia formed as they aged but did not produce sclerotia. Based on nuclear fluorescence staining with 1 µg·mL-1 4'-6-diamidino-2-phenylindole as described by Ahvenniemi et al. (2009), there were two nuclei per hyphal cell for all the 12 isolates. Moreover, the sequences of internal transcribed spacer region of ribosomal DNA (rDNA-ITS) of all the 12 isolates were amplified using the primers ITS1/ITS4 (White et al. 1990). For identical sequences, only the rDNA-ITS sequence (674 bp) of Dcp-19 has been deposited in GenBank (accession no. ON004932) and BLASTn analyses showed 100% homology with Rhizoctonia AG-K (MF070696). Maximum likelihood phylogenetic analysis further confirmed the identification. Healthy C. pilosula plants grown for two years in hillside fields were transplanted into sterile soil for pathogenicity testing. And the 12 isolates were all done in this test. Sterilized wheat seeds were placed on a 2-day-old colony of the isolate and incubated for 7 days. One fungus-infested seed was placed at the base of the stem and covered with sterilized soil. Control plants were inoculated with sterilized wheat seeds. Tests were performed on three plants for each isolate. The experiment was repeated twice. All the plants were cultivated at 22 °C and 50% relative humidity. After three weeks, the basal stems of the control plants were still healthy and did not have lesions, but the treated plants exhibited sunken brown canker lesions. The mean disease incidence of all the 12 isolates was 58.33%. The AG-K isolates re-isolated from the lesions of treated plants were confirmed by the morphological and molecular characteristics mentioned above, fulfilling Koch's postulates. To our knowledge, this is the first report of stem canker on C. pilosula caused by binucleate Rhizoctonia AG-K in China.

Anastomosis Groups of Rhizoctonia solani and Binucleate Rhizoctonia Associated with Potatoes in Idaho.

A survey of the relative incidence of anastomosis groups (AGs) of Rhizoctonia spp. associated with potato disease was conducted in Idaho, the leading potato producing state in the U.S.A. In total, 169 isolates of Rhizoctonia solani and seven binucleate Rhizoctonia (BNR) isolates were recovered from diseased potato plants. The AG of each isolate was determined through real-time PCR assays for AG 3-PT and phylogenetic analysis of the internal transcribed spacer region of ribosomal DNA. AG 3-PT was the predominant AG, accounting for 85% of isolates recovered, followed by AG 2-1 (5.7%) and AG 4 HG-II (4.5%). Two different subsets of AG 2-1 isolates were recovered (subset 2 and 3). Three isolates each of AG A and AG K were recovered, as well as one isolate each of AG 5 and AG W. An experiment carried out under greenhouse conditions with representative isolates of the different AGs recovered from Idaho potatoes showed differences in aggressiveness between AGs to potato stems, with AG 3-PT being the most aggressive followed by an isolate of AG 2-1 (subset 3). The three BNR isolates representative of AG A, AG K, and AG W appeared to be less aggressive to potato stems than the R. solani isolates except for the AG 2-1 (subset 2) isolate. This is the first comprehensive study of the relative incidences of Rhizoctonia species associated with Idaho potatoes and the first study to report the presence of BNR AG W outside of China.

Identification and Characterization of Sphaerulina vaccinii sp. nov. as the Cause of Leaf Spot and Stem Canker in Lowbush Blueberry and Its Epidemiology.

Septoria leaf spot and stem canker is an important disease of lowbush blueberry, but the causal pathogen has not been accurately identified. Based on sequence analysis of the internal transcribed spacer, translation elongation factor 1 alpha, RNA polymerase II second largest subunit, 28S nuclear ribosomal DNA gene, and ß-tubulin genes, the pathogen aligns closely with the genus Sphaerulina. The phylogenetic analyses based on these loci demonstrate that while the pathogen is closely related to the species Sphaerulina amelanchier, it is sufficiently distinct to warrant a new species designation. No ascomata of the teleomorph were found; however, ascospores recovered from leaves fit, morphologically, with the genus Mycosphaerella. The morphological data also support a new species designation. Based on the host that this pathogen infects, we propose the name as Sphaerulina vaccinii and the disease as Sphaerulina leaf spot and stem canker. Under field conditions, it appears that initial inoculum originates from pycnidia on overwintered leaves and stem lesions (cankers) on fruiting stems. More than 90% of the initial inoculum was released during the flowering period from late May through June. Leaf spots began to appear in early June and disease severity increased in a linear manner over time. Secondary inoculum production from diseased foliage was minimal and not considered important epidemiologically. Defoliation resulting from disease began in early July and increased in a nonlinear manner thereafter. Manual defoliation of blueberry stems at various times prior to harvest showed the substantial extent to which premature defoliation by this disease can affect yield. Stem lesions were also shown to have an impact on yield, even though stems were not killed.

First Report of Paramyrothecium foliicola Causing Stem Canker of Cucumber (Cucumis sativus L.) seedlings in China.

In April 2017, stem canker symptoms were observed on cucumber seedings grown in a greenhouse (0.1 ha) in Wuqing District, Tianjin(39°34' N; 117°07' E), China. Initially, the observed symptoms included small necrotic lesions of a light brown color on the stem base. These lesions subsequently spread and turned a darker brown. The leaves of the affected plants turned yellow and wilted. As the disease progressed the plants eventually died. Years of growing cucumbers and sufficient soil moisture in the greenhouse, might have led to a disease incidence of approximately 7%. Symptomatic tissue pieces were surface disinfested in 2% sodium hypochlorite for 60 s, rinsed three times in sterile water, and subsequently plated on potato dextrose agar (PDA) incubated at 25°C . At three days of incubation, mycelia appeared, turned into white and floccose isolated colonies around the excised tissue, and developed olivaceous green concentric rings of sporodochia in the following days. A total of 20 isolates with similar morphology were examined. Five single-spore isolates of isolates designated TJWQPF1-TJWQPF5 were obtained and maintained on PDA at 25°C. Hyaline, cylindrical conidiogenous cells measuring 9.53 to 16.51 × 1.51 to 2.49 µm (n=50) developed in whorls of three to six on terminal branches. Conidia were single-celled, hyaline, and rod-shaped with rounded ends. Conidia size averaged 5.07 - 7.15 × 1.13 - 2.32 µm (n=50). These characteristics are similar to the morphology of Paramyrothecium foliicola (Lombard et al. 2016). To further identify the isolate TJWQPF1, genomic DNA was extracted and the internal transcribed spacer (ITS, White et al. 1990), ß-tubulin (tub2, Glass & Donaldson 1995), RNA polymerase II largest subunit (rpb2, O'Donnell et al. 2007) and calmodulin (cmdA, Carbone & Kohn 1999; Groenewald et al. 2013) genes regions were amplified using the primer pairs ITS4/ITS5, Bt2a /Bt2b, RPB2-5F2 /RPB2-7cR, CAL-228F /CAL2Rd , respectively. All sequences were obtained and deposited in GenBank. BLAST searches of the NCBI database revealed that the ITS ( MW092223 ), tub2( MW110635 ) , rpb2 ( MW110637 ) and cmdA ( MW110636 ) sequences of the isolate TJWQPF1 were 100% identical to Paramyrothecium foliicola (GenBank accession numbers MT415351 and MT415352 for ITS sequences; MT415353 for tub2 sequences; MN398028-MN398043 for rpb2 sequences; MN593698- MN593713 for cmdA sequences). We also sequenced the other four single isolates and identified them as P. foliicola. Pathogenicity tests were conducted and repeated three times. Briefly, ten healthy 45-day-old cucumber seedlings (cultivar:Jinlv No.3) were inoculated with 100 µL of conidial suspension of P. foliicola (5×105 conidia per ml). Inoculum was applied to the stem with a syringe. Three healthy cucumber seedlings had 100 µL sterile water injected into the stem to serve as controls. All treated plants were incubated in a climate-controlled growth chamber at 25℃ (90% humidity, 12:12 h light:dark). Symptoms appeared on all inoculated plants after 7 days. In contrast, control seedlings exhibited no symptoms. The fungus was re-isolated from symptomatic tissues and re-identified to be P. foliicola, thereby fulfilling Koch's postulates. To our knowledge, this is the first known instance of P. foliicola inducing stem canker on cucumber plants in China. Stem canker caused by P. foliicola could pose a threat to cucumber production in China. Our results also provide a basis to monitor and manage this potential disease.

First Report of Stem Canker of Almond Trees Caused by Fusarium solani in Greece.

Almond (Prunus dulcis) is an important crop for Greece grown on 15.130 ha in 2019. In September 2019, a severe stem canker disease was observed in 6-year-old trees of cv Marta grafted on the rootstock 'F675C14', in a new almond grove of cvs Marta, Soleta, Antonela, Belona and Laurete, in Vlachiana, Heraklion, Crete, Greece. Only cv Marta trees were affected. Diseased trees exhibited cankers on trunks and branches with pale yellow to red-colored gum excreting from cankers, yellowing, leaf fall, twig and branch dieback, bark and wood tissue discoloration. Severely affected trees were killed. A Fusarium-like fungus was consistently isolated from symptomatic wood tissue previously surface-disinfested with 95% ethanol, on acidified potato dextrose agar (APDA). Emerging colonies were transferred to new PDA and the growth rate of the fungus was 7.86 mm/day at 24 °C in the dark. The abundant aerial mycelium was initially white, turning into pale orange in the centre after 7 days of growth on PDA. Microscopic observations revealed hyaline conidiophores measuring 26.74 ± 20.44 µm in length, developing microconidia 5.00 to 9.50 × 2.50 to 4.75 µm (average 6.64 × 3.50 µm) and macroconidia 10.00 to 23.25 × 3.75 to 5.50 µm (average 16.42 × 4.50 µm) in size. DNA from one representative single-spore isolate (code KOUB.AM.VR1) was extracted and the internal transcribed spacer region (ITS) of ribosomal DNA and translation elongation factor 1-alpha (EF 1-a) genes were amplified using the primer pairs ITS1/ITS4 (White et al. 1990) and EF1-F/EF2-R (O'Donnell et al. 1998), respectively. The PCR products were sequenced and deposited in GenBank (accession Nos. MW547397 and MW554492). Based on morphological characteristics (Leslie and Summerell 2006) and a BLAST search with 100.00% and 99.38% identity to published F. solani ITS and EF 1-a sequences in GenBank (KX034335.1, DQ247636.1) the fungus was identified as F. solani. Eight 3-year-old almond trees of cv. Marta were artificially inoculated in March 2020 by making a 6.0-mm-diameter hole into the trunk, inserting a 6-mm-diameter mycelial disc taken from a 10-day-old PDA culture, sealing the hole with cellophane membrane and covering with adhesive paper tape. Another eight trees of the same cultivar were mock-inoculated with sterilized PDA discs and served as controls. Potted trees were kept under ambient conditions. One month post inoculation, yellow gum was evident excreting around the inoculation point in F. solani-treated trees but not in the controls. Seven months post inoculation, longitudinal and transverse sections of inoculated trunks revealed internal and external symptoms similar to those observed under natural infection conditions and F. solani was steadily re-isolated from symptomatic wood tissue and identified by colony morphology. Neither symptoms nor positive isolations were observed in control trunks. Pathogenicity tests were repeated twice. Fusarium solani has been reported as the causal agent of stem canker or wood decay diseases in several woody hosts including bitternut hickory, black walnut, mulberry and pistachio trees (Crespo et al. 2019; Markakis et al. 2017; Park and Juzwik 2012; Tisserat 1987). To the best of our knowledge, this is the first worldwide report of stem canker caused by F. solani on almond tree. This disease could potentially be an increasing problem in almond growing areas and result in severe crop losses. Hence, effective management practices should be investigated and applied.

First Report of Diaporthe phaseolorum Causing Stem Canker of Hemp (Cannabis sativa).

Hemp is an annual herbaceous plant that is used for its fiber and oil in a variety of commercial and industrial products. In Florida, it is currently being explored as a new specialty crop. During a field trial from October to January 2019 in Wimauma, FL, a stem canker was observed on up to 60% of three-month-old plants of 'Eletta Campana', 'Carmagnola Selezionata', and 'Tygra'. Symptoms started on the main stems with light-to-dark brown lesions of different sizes and shapes. Over time, the lesions coalesced into large necrotic areas and bore pycnidia. Isolations were made from diseased stem tissues on General Isolation medium (Amiri et al. 2018) after surface disinfestation (Marin et al. 2020). The plates were placed in a growth chamber at 25°C under a 12/12 photoperiod. A fungus with white, floccose, aerial mycelium and pycnidia producing alpha and beta conidia was consistently isolated. Three single spore isolates were chosen for identification and pathogenicity tests. Pycnidia on PDA were globose to irregular and ranged from 170 to 250 µm long (210 ± 2.5, n = 50) and 140 to 220 µm wide (180 ± 2.7, n = 50). The alpha conidia were unicellular, hyaline, ellipsoidal to fusiform and ranged from 5.3 to 7.7 µm long (6.5 ± 1.6, n = 50) and 1.5 to 4.6 µm wide (2.8 ± 1.8, n = 50). The beta conidia were hyaline, elongated, filiform, straight or curved and ranged from 10.2 to 17.7 µm long (16.1 ± 2.2, n = 50) and 0.5 to 1.8 µm wide (0.8 ± 0.2, n = 50). Perithecia were not observed. Based on morphological features, the fungus was similar to anamorphs of Diaporthe spp. (Santos et al. 2011; Udayanga et al. 2015). DNA from the same three isolates was extracted using the FastDNA kit, and the ribosomal internal transcribed spacer (ITS), ß-tubulin (TUB), and calmodulin (CAL) regions were amplified following Udayanga et al. (2014), and Sanger sequenced by Genewiz. Sequences were deposited in GenBank (accession no. MT497039 to MT497047 for ITS, TUB, and CAL). BLASTn searches revealed isolates 20-58, 20-59, and 20-60 were 96.34% identical to the epitype isolate D. phaseolorum AR4203 for ITS (KJ590738.1, 527 bp out of 547 bp), 100% for TUB (KJ610893.1, 459 bp out of 459 bp), and 100% for CAL (KJ612135.1, 522 bp out of 522 bp) (Udayanga et al. 2015). Their identity was confirmed by phylogenetic analyses using maximum likelihood and Bayesian inference methods. To complete Koch's postulates, pycnidia of the same three isolates were harvested and crushed in 2 mL Eppendorf tubes containing 0.01% Tween 20. Conidia suspensions were adjusted to 106 spores/mL. Three 5-week-old potted plants of 'Eletta Campana' and 'Carmagnola Selezionata' per isolate were inoculated using a 1 mL syringe with a needle by injecting 200 µL of the suspension into the stem. Plants were placed inside clear plastic bags for 48 h and maintained in the greenhouse. Control plants were injected with sterile deionized water and kept under the same conditions. The pathogenicity test was repeated once. Four weeks after inoculation, inoculated plants developed stem cankers from which the same pathogen was isolated, whereas controls remained healthy. To our knowledge, this is the first report of D. phaseolorum causing stem canker on hemp. This pathogen has been reported causing canker on sunflower and Phaseolus spp. (Gomzhina and Gannibal 2018; Udayanga et al. 2015; Vrandecic et al. 2009). This discovery may help shape future research into disease epidemiology and management for a crop in which very limited disease information is available at the moment.

Identification of resistance loci in Chinese and Canadian canola/rapeseed varieties against Leptosphaeria maculans based on genome-wide association studies.

BACKGROUND: The fungal pathogen Leptosphaeria maculans (Lm). causes blackleg disease on canola/rapeseed in many parts of the world. It is important to use resistant cultivars to manage the disease and minimize yield losses. In this study, twenty-two Lm isolates were used to identify resistance genes in a collection of 243 canola/rapeseed (Brassica napus L.) accessions from Canada and China. These Lm isolates carry different compliments of avirulence genes, and the investigation was based on a genome-wide association study (GWAS) and genotype-by-sequencing (GBS). RESULTS: Using the CROP-SNP pipeline, a total of 81,471 variants, including 78,632 SNPs and 2839 InDels, were identified. The GWAS was performed using TASSEL 5.0 with GLM + Q model. Thirty-two and 13 SNPs were identified from the Canadian and Chinese accessions, respectively, tightly associated with blackleg resistance with P values < 1 × 10- 4. These SNP loci were distributed on chromosomes A03, A05, A08, A09, C01, C04, C05, and C07, with the majority of them on A08 followed by A09 and A03. The significant SNPs identified on A08 were all located in a 2010-kb region and associated with resistance to 12 of the 22 Lm isolates. Furthermore, 25 resistance gene analogues (RGAs) were identified in these regions, including two nucleotide binding site (NBS) domain proteins, fourteen RLKs, three RLPs and six TM-CCs. These RGAs can be the potential candidate genes for blackleg resistance. CONCLUSION: This study provides insights into potentially new genomic regions for discovery of additional blackleg resistance genes. The identified regions associated with blackleg resistance in the germplasm collection may also contribute directly to the development of canola varieties with novel resistance genes against blackleg of canola.

Stem Canker on Cyclocarya paliurus Is Caused by Botryosphaeria dothidea.

Cyclocarya paliurus, an important endangered plant in China, has considerable medicinal, timber, and horticultural value. However, little is known about diseases that affect its health. In recent years, stem canker diseases on C. paliurus have been observed frequently in newly established nurseries in Jiangsu Province, China. Symptomatic trees showed elliptical, sunken lesions on the bark, with internal discoloration, leading to enlarging cankers with delineated margins. Pathogenicity tests with fungi isolated from symptomatic samples reproduced typical canker symptoms on both detached branches and potted plants of C. paliurus. Moreover, conidia from pycnidia of isolate ZB-23 could also cause stem canker on C. paliurus. Through combined morphological observation and DNA sequences of ITS region, ß-tubulin, and translation elongation factor 1-α genes, the pathogen was identified as Botryosphaeria dothidea. Multigene maximum likelihood and maximum parsimony phylogenetic analyses further supported the identification of the pathogen. To our knowledge, this is the first report of B. dothidea causing stem canker on C. paliurus in China.

Quantitative PCR Assays Developed for Diaporthe helianthi and Diaporthe gulyae for Phomopsis Stem Canker Diagnosis and Germplasm Screening in Sunflower (Helianthus annuus).

Phomopsis stem canker of sunflower is caused by two fungal pathogens, Diaporthe helianthi and Diaporthe gulyae, in the United States. In this study, two quantitative PCR (qPCR) assays were developed to detect and quantify D. helianthi and D. gulyae in sunflower. The two assays differentiated the two fungi from each other, other species of the genus Diaporthe, and pathogens, and they have high efficiency (>90%). The qPCR assays detected the two pathogens on plant samples exhibiting Phomopsis stem canker symptoms sampled from commercial sunflower fields in Minnesota, Nebraska, North Dakota, and South Dakota. Furthermore, the assays were used to screen cultivated sunflower accessions for resistance to D. helianthi and D. gulyae. The disease severity index (DSI) of the accessions significantly correlated (P < 0.0001) with the amount of pathogen DNA from the qPCR assays. The qPCR assays identified PI664232 and PI561918 to be significantly less susceptible (P ≤ 0.05) to D. helianthi and D. gulyae, respectively, when compared with the susceptible check cultivar HA 288, and this was in agreement with the DSI. These results suggest that the qPCR assays for D. helianthi and D. gulyae can be used as a reliable tool to diagnose Phomopsis stem canker and screen sunflower germplasm for disease resistance.

Molecular Characterization and Disease Control of Stem Canker on Royal Poinciana (Delonix regia) Caused by Neoscytalidium dimidiatum in the United Arab Emirates.

In the United Arab Emirates (UAE), royal poinciana (Delonix regia) trees suffer from stem canker disease. Symptoms of stem canker can be characterized by branch and leaf dryness, bark lesions, discoloration of xylem tissues, longitudinal wood necrosis and extensive gumming. General dieback signs were also observed leading to complete defoliation of leaves and ultimately death of trees in advanced stages. The fungus, Neoscytalidium dimidiatum DSM 109897, was consistently recovered from diseased royal poinciana tissues; this was confirmed by the molecular, structural and morphological studies. Phylogenetic analyses of the translation elongation factor 1-a (TEF1-α) of N. dimidiatum from the UAE with reference specimens of Botryosphaeriaceae family validated the identity of the pathogen. To manage the disease, the chemical fungicides, Protifert®, Cidely® Top and Amistrar® Top, significantly inhibited mycelial growth and reduced conidial numbers of N. dimidiatum in laboratory and greenhouse experiments. The described "apple bioassay" is an innovative approach that can be useful when performing fungicide treatment studies. Under field conditions, Cidely® Top proved to be the most effective fungicide against N. dimidiatum among all tested treatments. Our data suggest that the causal agent of stem canker disease on royal poinciana in the UAE is N. dimidiatum.