Canker Pathogen Research Articles

Secreted Effector Proteins of Poplar Leaf Spot and Stem Canker Pathogen Sphaerulina musiva Manipulate Plant Immunity and Contribute to Virulence in Diverse Ways.

Fungal effectors play critical roles in manipulating plant immune responses and promoting colonization. Sphaerulina musiva is a heterothallic ascomycete fungus that causes Septoria leaf spot and stem canker disease in poplar (Populus spp.) plantations. This disease can result in premature defoliation, branch and stem breakage, increased mortality, and plantation failure. However, little is known about the interaction between S. musiva and poplar. Previous work predicted 142 candidate secreted effector proteins in S. musiva (SmCSEPs), 19 of which were selected for further functional characterization in this study. SmCSEP3 induced plant cell death in Nicotiana benthamiana, while 8 out of 19 tested SmCSEPs suppressed cell death. The signal peptides of these eight SmCSEPs exhibited secretory activity in a yeast signal sequence trap assay. Confocal microscopy revealed that four of these eight SmCSEPs target both the cytoplasm and the nucleus, whereas four predominantly localize to discrete punctate structures. Pathogen challenge assays in N. benthamiana demonstrated that the transient expression of six SmCSEPs promoted Fusarium proliferatum infection. The expression of these six SmCSEP genes were induced during infection. SmCSEP2, SmCSEP13, and SmCSEP25 suppressed chitin-triggered reactive oxygen species burst and callose deposition in N. benthamiana. The candidate secreted effector proteins of S. musiva target multiple compartments in the plant cell and modulate different pattern-triggered immunity pathways. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

Seasonal Susceptibility of Sweet Cherry Pruning Wounds to Calosphaeria pulchella, Cytospora sorbicola, and Eutypa lata.

Fungal canker pathogens commonly infect trees at pruning wounds leading to branch dieback and loss of productivity in sweet cherry orchards. However, the seasonal susceptibility of sweet cherry pruning wounds to Calosphaeria pulchella, Cytospora sorbicola, and Eutypa lata is not well understood. This study compared the susceptibility of sweet cherry pruning wounds made during the dormant season (January) and the postharvest season (late May to June) to infection by main canker pathogens in California. Field trials were conducted in three cherry orchards and trees were pruned at the different periods over 2 years. Fresh pruning wounds were inoculated with spores of each pathogen, and pathogen recovery was assessed through microbiological isolations at 3 to 4 months after inoculations. Pruning wounds made in late May and June resulted in significantly higher infection by Cal. pulchella compared to pruning wounds made in January. Pruning wounds made during both seasons were generally equally susceptible to Cyt. sorbicola and E. lata infections. However, there was one orchard where dormant pruning wounds were more susceptible to infection by E. lata and there was one particularly cold winter where Cyt. sorbicola did not infect pruning wounds. Overall, our findings suggest that Cal. pulchella infections of cherry pruning wounds are more likely to occur during periods of warm temperatures such as late spring and early summer. However, infections by Cyt. sorbicola and E. lata can occur year-round if inoculum is present and if winter temperatures are not abnormally low for California. Finally, our results suggest that the emergence of Cal. pulchella as a major canker pathogen of sweet cherry in California may be the result of a shift from dormant to after-harvest pruning of sweet cherry trees.

Fungi associated with dieback of apple trees to be excavated and the assessment of Diplodia seriata fruiting structures on chipped apple wood used for mulch

Excavated apple trees as part of orchard renewal are often chipped and used as mulch in younger orchards. Therefore, the extent of fungal pathogens associated with dieback in old apple orchards to be excavated was investigated, with an emphasis on Diplodia seriata, an important canker pathogen of Malus domestica in South Africa. Trees showing symptoms of dieback were sampled from 14 orchards in the Grabouw, Vyeboom and Kouebokkeveld apple-producing areas in South Africa. Isolations were made from surface sterilised internal tissue of dieback samples. For the detection of D. seriata inoculum from chipped apple wood pieces, samples were collected in October 2020 and from February to April 2021 of orchards in the Grabouw (2 orchards) and Vyeboom (1 orchard) production regions. Fungi associated with canker and wood rot symptoms were isolated from 118 of the 144 trees showing symptoms of dieback. Eutypa lata was most often isolated (from 18.6 % of trees) followed by D. seriata (9.3 %). The predominant wood rot pathogen associated with the dieback of apple trees was Trametes versicolor (from 33.1 % of trees). Visual inspections of mulch wood pieces indicated the presence of D. seriata pycnidia and or conidia from material assessed between October 2020 and April 2021. Viable D. seriata cultures were obtained from 87 % of the mulch wood pieces investigated. Diplodia seriata remained present and viable for a period of 20 months on the wood chips. This study showed that apple trees to be excavated and used for mulch, harbor important canker and wood rot pathogens. The presence of viable D. seriata inoculum on apple tree wood chips used for mulch in younger orchards illustrates that there is a risk involved in using wood chips made from old fruit trees. Infected tree parts and entire dead trees should thus be removed before the chipping of older apple trees to be used for mulch.

Co-inoculation timing affects the interspecific interactions between phoma stem canker pathogens Leptosphaeria maculans and Leptosphaeria biglobosa.

Phoma stem canker is an economically important disease of oilseed rape, caused by two co-existing fungal pathogen species, Leptosphaeria maculans (Plenodomus lingam) and Leptosphaeria biglobosa (Plenodomus biglobosus). Leptosphaeria maculans produces a phytotoxin called sirodesmin PL. Our previous work showed that L. biglobosa has an antagonistic effect on the production of sirodesmin PL if it is simultaneously co-inoculated with L. maculans. However, the effects of sequential co-inoculation on interspecific interactions between the two pathogens are not understood. The interactions between L. maculans and L. biglobosa were investigated in liquid culture by inoculation with L. maculans first, followed by L. biglobosa sequentially at 1, 3, 5 or 7 days later and vice versa; the controls were inoculated with L. maculans only, L. biglobosa only, or L. maculans and L. biglobosa simultaneously. The results showed that L. biglobosa inhibited the growth of L. maculans, the production of both sirodesmin PL and its precursors if L. biglobosa was inoculated before, or simultaneously with, L. maculans. However, the antagonistic effects of L. biglobosa were lost if it was co-inoculated 5 or 7 days after L. maculans. For the first time, the results of this study provided evidence that the timing when L. maculans and L. biglobosa meet significantly influences the outcome of the interspecific competition between them. Leptosphaeria biglobosa can inhibit the production of sirodesmin PL and the growth of L. maculans if it is inoculated before L. maculans or less than 3 days after L. maculans in liquid culture. There is a need to further investigate the timing of co-inoculation on interactions between L. maculans and L. biglobosa in their host plants for improving the control of phoma stem canker. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Identification and genetic characterization of Pseudomonas syringae pv. actinidiae from kiwifruit in Sichuan, China.

Pseudomonas syringae pv. actinidiae (Psa) causes kiwifruit bacterial canker and poses a major threat to the kiwifruit industry. This study aimed to investigate the genetic characteristics of the Psa population from kiwifruit in Sichuan, China. Sixty-seven isolates obtained from diseased plants were characterized using morphological features, multiplex-PCR and multilocus sequence analysis (MLSA). The isolates exhibited the typical colony morphology of Psa. Multiplex PCR amplification identified every isolate as Psa biovar 3. MLSA analysis of the three housekeeping genes gapA, gyrB, and pfk, revealed that the reference strains of the five described biovars were clearly distinguished by a combined phylogenetic tree, and all the tested isolates clustered with the reference strains of Psa biovar 3. Through a phylogenetic tree constructed from a single gene, it was found that pkf gene alone could distinguish biovar 3 from the other biovars. Furthermore, all Psa isolates analyzed by BOX-A1R-based repetitive extragenic palindromic (BOX)-PCR and enterobacterial repetitive intergenic consensus (ERIC)-PCR clustered into four groups. The clustering results of BOX- and ERIC-PCR indicated that group III had the largest number of isolates, accounting for 56.72% and 61.19% of all sixty-seven isolates, respectively, and the two characterization methods were similar and complementary. The results of this study revealed that the genomes of Psa isolates from Sichuan had rich genetic diversity, but no obvious correlation was found between clustering and geographical region. This research provides novel methodologies for rapidly detecting kiwifruit bacterial canker pathogen and a molecular differentiation at genetic level of Psa biovars diversity in China.

Whole genome sequencing and analysis of multiple isolates of Ceratocystis destructans, the causal agent of Ceratocystis canker of almond in California

Ceratocystis canker caused by Ceratocystis destructans is a severe disease of almond, reducing the longevity and productivity of infected trees. Once the disease has established in an individual tree, there is no cure, and management efforts are often limited to removing the infected area of cankers. In this study, we present the genome assemblies of five C. destructans isolates isolated from symptomatic almond trees. The genomes were assembled into a genome size of 27.2 ± 0.9 Mbp with an average of 6924 ± 135 protein-coding genes and an average GC content of 48.8 ± 0.02%. We concentrated our efforts on identifying putative virulence factors of canker pathogens. Analysis of the secreted carbohydrate-active enzymes showed that the genomes harbored 83.4 ± 1.8 secreted CAZymes. The secreted CAZymes covered all the known categories of CAZymes. AntiSMASH revealed that the genomes had at least 7 biosynthetic gene clusters, with one of the non-ribosomal peptide synthases encoding dimethylcoprogen, a conserved virulence determinant of plant pathogenic ascomycetes. From the predicted proteome, we also annotated cytochrome P450 monooxygenases, and transporters, these are well-established virulence determinants of canker pathogens. Moreover, we managed to identify 57.4 ± 2.1 putative effector proteins. Gene Ontology (GO) annotation was applied to compare gene content with two closely related species C. fimbriata, and C. albifundus. This study provides the first genome assemblies for C. destructans, expanding genomic resources for an important almond canker pathogen. The acquired knowledge provides a foundation for further advanced studies, such as molecular interactions with the host, which is critical for breeding for resistance.

First Report of Cytospora azerbaijanica Causing Cytospora Canker and Shoot Dieback on Peach (Prunus persica) in California, U.S.A.

Peaches (Prunus persica L.) are an important crop in the United States with California leading the nation in peach production, with approximately 505,000 tons valued at $378.3 million (USDA National Agricultural Statistics Service, 2021, https://www.nass.usda.gov/). From April to July 2022, symptoms of branch and scaffold canker as well as shoot dieback were observed in three peach (cvs. Loadel, Late Ross and Starn) orchards located in San Joaquin County, California. Samples were collected from about 12 trees for each cultivar. Fast-growing, white, flat colonies were consistently isolated from active cankers on acidified potato dextrose agar (APDA) following the method described by (Lawrence et al. 2017). Pure fungal cultures were obtained by transferring single hyphal tips onto new APDA Petri plates. A total of 22 isolates were obtained. Each fungal isolate was recovered from a single diseased branch (40 to 55% recovery). All isolates in this study shared similar morphological characteristics. Fungal colonies were fast-growing with relatively even but slightly dentate margin, flat with white to off-white mycelium that turned vinaceous buff to pale greyish sepia (Rayner 1970) with age. Black, globose, ostiolated pycnidia, 0.8-(1.3)-2.2 mm diameter, with brownish surface hyphae formed on peach wood embedded in PDA after approximately three weeks and exudated buff-colored mucilage. Pycnidia were both solitary and aggregated and had multiple internal locules sharing invaginated walls. Conidiogenous cells were hyaline, smooth-walled, septate, tapering towards the apex, 13-(18.2)-25.1 × 0.8-(1.3)-1.9 µm (n = 40). Conidia were hyaline, allantoid, smooth, aseptate, 5.5-(6.3)-7.1 × 1.4-(1.9)-2.3 µm (n = 40). Genomic DNA was extracted and sequences of the internal transcribed spacer region (ITS) using ITS5/ITS4 universal primers, translation elongation factor 1α gene (TEF) using primers EF1-728F/EF1-986R, second largest subunit of RNA polymerase II (RPB2) using primers RPB2-5F2/fRPB2-7cR, and actin gene region (ACT) using primers ACT-512F/ACT-783R were obtained and compared with sequences available in GenBank (Lawrence et al. 2018; Hanifeh et al. 2022). Isolates were identified as Cytospora azerbaijanica following DNA sequencing and morphological identification. Consensus sequences of the four genes of two representative isolates (SJC-66 and SJC-69) were deposited into GenBank database (ITS: OQ060581 and OQ060582; ACT: OQ082292, OQ082295; TEF: OQ082290 and OQ082293; RPB2: OQ082291 and OQ082294). The Basic Local Alignment Search Tool (BLAST) indicated that the sequenced RPB2 genes of isolates (SJC-66 and SJC-69) were at least 99% identical to that of Cytospora sp. strain shd47 (Accession: MW824360) covering at least 85% of the sequences. The actin genes from our isolates were at least 97.85% identical to that of Cytospora sp. strain shd47 (Accession: MZ014513), covering 100% of the sequences. The translation elongation factor gene from isolates (SJC-66 and SJC-69) was at least 96.4% identical to that of Cytospora sp. strain shd166 (Accession: OM372512), covering 100% of the query. Those top hit strains belong to C. azerbaijanica, recently reported by Hanifeh et al. (2022). Pathogenicity tests were performed by inoculating eight wounded, 2- to 3-year-old healthy branches on each of eight 7-year-old peach trees, cvs. Loadel, Late Ross and Starn, using 5-mm-diameter mycelium plugs collected from the margin of an actively growing fungal colony on APDA. Controls were mock-inoculated with sterile agar plugs. Inoculation sites were covered with petroleum jelly and wrapped with Parafilm to keep moisture. The experiment was performed twice. After four months, inoculation tests resulted in vascular discoloration (canker) above and below the inoculation sites (average necrosis length of 114.1 mm). Cytospora azerbaijanica was re-isolated from all infected branches (70 to 100% recovery) completing Koch's postulates. Controls remained symptomless and no fungi were isolated from the slightly discolored tissue. Cytospora species are destructive canker and dieback pathogens of numerous woody hosts worldwide. Recently, C. azerbaijanica was reported in causing canker disease of apple trees in Iran (Hanifeh et al. 2022). To our knowledge, this is the first report of C. azerbaijanica causing canker and shoot dieback of peach trees in the United States and worldwide. These findings will aid towards a better understanding of genetic diversity and host range of C. azerbaijanica.

The role of stress factors in severity of Cytospora plurivora in greenhouse and field plantings of 13 peach (Prunus persica) cultivars.

Understanding the host-pathogen-environmental interactions in a pathosystem is essential for management of diseases and diminished crop yields. Abiotic stressors such as cold damage, water deficit, and high pH soils can be major limiting factors to tree fruit production. Along with decreased yields, these abiotic factors can have direct implications for disease severity within orchards. Cytospora plurivora is a ubiquitous fungal canker pathogen in western Colorado, USA and is a major focus in integrated pest management strategies. This research evaluated the influence of biotic and abiotic stress factors on peach tree health. Thirteen peach cultivars were placed under abiotic stress and inoculated with C. plurivora in greenhouse and field conditions. Under deficit irrigation, C. plurivora infections were significantly larger and more severe in both the greenhouse and field trials when compared with those under the full-irrigation controls. In controlled greenhouse conditions, a positive correlation between lesion size and water potential was evident, but no trend of cultivar tolerance was observed. Furthermore, increase in irrigation water pH, through additions of sodium carbonate and bicarbonate, in the greenhouse trials resulted in decreased leaf water potentials and increased pathogen necrotic tissue volumes (mm3). In field trials, there was no positive relationship between lesion size and water potential; trees with the most negative water potentials had the smallest lesions sizes that did not correspond to cultivar, suggesting that other abiotic or biotic factors may be shielding water stressed trees from increased pathogen aggression. This research highlights the importance of proper irrigation and soil pH management as tools for the management of Cytospora canker in peach orchards.

Comparison of assembly platforms for the assembly of the nuclear genome of Trichoderma harzianum strain PAR3

BackgroundTrichoderma is a diverse genus of fungi that includes several species that possess biotechnological and agricultural applications, including the biocontrol of pathogenic fungi and nematodes. The mitochondrial genome of a putative strain of Trichoderma harzianum called PAR3 was analyzed after isolation from the roots of Scarlet Royal grapevine scion grafted to Freedom rootstock, located in a grapevine vineyard in Parlier, CA, USA. Here, we report the sequencing, comparative assembly, and annotation of the nuclear genome of PAR3 and confirm its identification as a strain of T. harzianum. We subsequently compared the genes found in T. harzianum PAR3 to other known T. harzianum strains. Assembly of Illumina and/or Oxford Nanopore reads by the popular long-read assemblers, Flye and Canu, and the hybrid assemblers, SPAdes and MaSuRCA, was performed and the quality of the resulting assemblies were compared to ascertain which assembler generated the highest quality draft genome assembly.ResultsMaSuRCA produced the most complete and high-fidelity assembly yielding a nuclear genome of 40.7 Mb comprised of 112 scaffolds. Subsequent annotation of this assembly produced 12,074 gene models and 210 tRNAs. This included 221 genes that did not have equivalent genes in other T. harzainum strains. Phylogenetic analysis of ITS, rpb2, and tef1a sequences from PAR3 and established Trichoderma spp. showed that all three sequences from PAR3 possessed more than 99% identity to those of Trichoderma harzianum, confirming that PAR3 is an isolate of Trichoderma harzianum. We also found that comparison of gene models between T. harzianum PAR3 and other T. harzianum strains resulted in the identification of significant differences in gene type and number, with 221 unique genes identified in the PAR3 strain.ConclusionsThis study gives insight into the efficacy of several popular assembly platforms for assembly of fungal nuclear genomes, and found that the hybrid assembler, MaSuRCA, was the most effective program for genome assembly. The annotated draft nuclear genome and the identification of genes not found in other T. harzainum strains could be used to investigate the potential applications of T. harzianum PAR3 for biocontrol of grapevine fungal canker pathogens and as source of anti-microbial compounds.

Evaluation of Biological Control Agents for the Protection of Almond Pruning Wounds Against Infection by Fungal Canker Pathogens.

Fungal canker pathogens of almond initiate infection in trees primarily through pruning wounds. Biological control agents (BCAs) have the potential to provide long-term protection of pruning wounds by colonizing the wound surfaces and underlying tissues. Laboratory and field tests were performed to assess the efficacy of various commercial and experimental BCAs as wound protectants against almond canker pathogens. Four Trichoderma-based BCAs were evaluated using detached almond stems in the laboratory against the canker pathogens Cytospora plurivora, Eutypa lata, Neofusicoccum parvum, and Neoscytalidium dimidiatum. Results indicated that Trichoderma atroviride SC1 and T. paratroviride RTFT014 significantly reduced infections by all four pathogens. The abilities of these four BCAs to protect almond pruning wounds against E. lata and N.parvum were further evaluated in field trials using two almond cultivars and during two consecutive years. Both T. atroviride SC1 and T. paratroviride RTFT014 protected almond pruning wounds against E. lata and N. parvum as efficiently as thiophanate-methyl, the recommended fungicide for treatment of almond pruning wounds. Comparisons of different application timings of BCA in relation to pathogen inoculation revealed a significant improvement in wound protection when inoculations were conducted 7 days versus 24 h post-BCA application for N. parvum, but not for E. lata. T. atroviride SC1 and T. paratroviride RTFT014 are promising candidates for the preventive protection of almond pruning wounds and for inclusion in integrated pest management programs and organic almond production systems.

Field evaluation of biological wound treatments for the management of almond, cherry, and grapevine fungal canker diseases

The ascomycete fungi that cause wood canker diseases in perennial, horticultural crops are often generalist pathogens able to infect multiple host plants. These pathogens frequently infect pruning wounds, and the protection of such wounds with chemicals or biological control agents (BCAs) is an essential component of disease management. This study aimed at assessing four commercial BCAs for their ability to prevent almond, cherry, and grapevine pruning wound infections by three canker pathogens under field conditions for two growing seasons: Calosphaeria pulchella in cherry, Neofusicoccum parvum in almond and grapevine, and Eutypa lata in all three hosts. The two bacterial BCAs tested, Bacillus amyloliquefasciens MBI 600 and Streptomyces lydicus WYEC 108, performed poorly overall as infection rates in treated wounds were not significantly different than in the water-treated wounds for most pathogens in all crops. Trichoderma atroviride SC1 provided the best protection against E. lata and N. parvum in grapevine, achieving superior or equivalent protection than the standard conventional chemical thiophanate-methyl. Field trials also demonstrated that Clonostachys rosea J1446 provided satisfactory protection of pruning wounds against E. lata and C. pulchella in cherry and against E. lata and N. parvum in almond trees. The identification of effective BCAs for use in multiple crops and against multiple pathogens is a great step towards a sustainable management of fungal canker diseases by mitigating the negative impacts of chemical fungicides.

Field Resistance of American Sycamore ‘Davis’ to Canker Pathogens

Abstract American sycamores (Platanus occidentalisL.) are found in many ecosystems and planted in urban landscapes worldwide. The trees are highly susceptible to anthracnose and canker pathogens, causing leaf blight and branch dieback. On The Ohio State University campus in Columbus, Ohio, an American sycamore was observed to thrive among many symptomatic sycamores. The healthy tree, subsequently protected as cultivar ‘Davis,’ was vegetatively propagated and tested for field resistance to natural infection of canker pathogens compared to the wildtype. Incidence and severity of leaf necrosis, incidence of dieback, and tree death were evaluated for 2 consecutive seasons. The incidence of leaf necrosis was disconnected from the incidence of dieback and tree mortality, as little to no leaves were produced on the wildtype trees. By the end of the second season, 7 out of 12 wildtype trees were dead, while all 12 ‘Davis’ trees were alive. Several canker pathogens were recovered from both ‘Davis’ and the wildtype, includingApiognomonia plataniandDiaporthe eres. The latter had not been previously reported on American sycamore. Pathogenicity tests confirmed thatD. eresis indeed pathogenic on sycamores and also that ‘Davis’ is significantly more resistant than wildtype to canker development and should be preferred over the wildtype in the urban landscape.

A fungal microRNA-like RNA regulated effector promotes pathogen infection by targeting a host defense-related transcription factor.

Effectors play important roles in facilitating the infection of plant pathogenic fungi. However, the gene expression regulatory mechanism of effector genes, especially at the post-transcriptional level, is largely unknown. In this study, we uncovered the post-transcriptional regulation of an effector gene VmSP1 by a miRNA-like RNA (Vm-milR16) facilitating the infection of the apple tree Valsa canker pathogen Valsa mali. Genetic and molecular biological assays indicated that the expression of VmSP1 could be suppressed by Vm-milR16-mediated mRNA cleavage in a sequence-specific manner. During V. mali infection, Vm-milR16 was down-regulated, whereas VmSP1 was upregulated, which further indicated the regulation relationship. VmSP1 was further demonstrated to be a secreted protein and could suppress plant immunity. Deletion of VmSP1 did not affect the vegetative growth but significantly reduced the virulence of V. mali. Further study indicated that VmSP1 could interact with the transcription factor MdbHLH189 of apple. Transiently overexpression of MdbHLH189 enhanced host resistance to V. mali by enhancing the expression of apple defense-related genes, together with the increased callose deposition. Silencing of MdbHLH189 compromised host resistance to V. mali. Our findings uncovered the novel epigenetic regulation mechanism of a virulence-associated effector gene mediated by a fungal milRNA at the post-transcriptional level, and the results enriched the understanding of the function and action mechanism of effectors in tree pathogenic fungi.

Delivery of Apoplastic Extracellular Vesicles Encapsulating Green-Synthesized Silver Nanoparticles to Treat Citrus Canker.

The citrus canker pathogen Xanthomonas axonopodis has caused severe damage to citrus crops worldwide, resulting in significant economic losses for the citrus industry. To address this, a green synthesis method was used to develop silver nanoparticles with the leaf extract of Phyllanthus niruri (GS-AgNP-LEPN). This method replaces the need for toxic reagents, as the LEPN acts as a reducing and capping agent. To further enhance their effectiveness, the GS-AgNP-LEPN were encapsulated in extracellular vesicles (EVs), nanovesicles with a diameter of approximately 30-1000 nm naturally released from different sources, including plant and mammalian cells, and found in the apoplastic fluid (APF) of leaves. When compared to a regular antibiotic (ampicillin), the delivery of APF-EV-GS-AgNP-LEPN and GS-AgNP-LEPN to X. axonopodis pv. was shown to have more significant antimicrobial activity. Our analysis showed the presence of phyllanthin and nirurinetin in the LEPN and found evidence that both could be responsible for antimicrobial activity against X. axonopodis pv. Ferredoxin-NADP+ reductase (FAD-FNR) and the effector protein XopAI play a crucial role in the survival and virulence of X. axonopodis pv. Our molecular docking studies showed that nirurinetin could bind to FAD-FNR and XopAI with high binding energies (-10.32 kcal/mol and -6.13 kcal/mol, respectively) as compared to phyllanthin (-6.42 kcal/mol and -2.93 kcal/mol, respectively), which was also supported by the western blot experiment. We conclude that (a) the hybrid of APF-EV and GS-NP could be an effective treatment for citrus canker, and (b) it works via the nirurinetin-dependent inhibition of FAD-FNR and XopAI in X. axonopodis pv.

Leptosphaeria maculans isolates with variations in AvrLm1 and AvrLm4 effector genes induce differences in defence responses but not in resistance phenotypes in cultivars carrying the Rlm7 gene.

The phoma stem canker pathogen Leptosphaeria maculans is one of the most widespread and devastating pathogens of oilseed rape (Brassica napus) in the world. Pathogen colonization is stopped by an interaction of a pathogen Avr effector gene with the corresponding host resistance (R) gene. While molecular mechanisms of this gene-for-gene interaction are being elucidated, understanding of effector function remains limited. The purpose of this study was to determine the action of L.maculans effector (AvrLm) genes on incompatible interactions triggered by B.napus noncorresponding R (Rlm) genes. Specifically, effects of AvrLm4-7 and AvrLm1 on Rlm7-mediated resistance were studied. Although there was no major effect on symptom expression, induction of defence genes (e.g. PR1) and accumulation of reactive oxygen species was reduced when B.napus cv. Excel carrying Rlm7 was challenged with a L.maculans isolate containing AvrLm1 and a point mutation in AvrLm4-7 (AvrLm1, avrLm4-AvrLm7) compared to an isolate lacking AvrLm1 (avrLm1, AvrLm4-AvrLm7). AvrLm7-containing isolates, isogenic for presence or absence of AvrLm1, elicited similar symptoms on hosts with or without Rlm7, confirming results obtained with more genetically diverse isolates. Careful phenotypic examination of isogenic L.maculans isolates and B.napus introgression lines demonstrated a lack of effect of AvrLm1 on Rlm7-mediated resistance despite an apparent alteration of the Rlm7-dependent defence response using more diverse fungal isolates with differences in AvrLm1 and AvrLm4. As deployment of Rlm7 resistance in crop cultivars increases, other effectors need to be monitored because they may alter the predominance of AvrLm7. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A LAMP Assay for Rapid Detection of the Pitch Canker Pathogen Fusarium circinatum.

The pine pitch canker pathogen Fusarium circinatum is endemic in the southeastern United States and Central America and represents an invasive threat globally. This ecologically adaptable fungus readily infects all parts of its pine hosts, leading to widespread mortality of nursery seedlings and decline in the health and productivity of forest stands. Because trees infected by F. circinatum can remain asymptomatic for long periods of time, accurate and rapid tools are needed for real-time diagnostics and surveillance at ports, in nurseries, and in plantations. To meet this need and to limit the spread and impact of the pathogen, we developed a molecular test using loop-mediated isothermal amplification (LAMP), a technology that allows for the rapid detection of pathogen DNA on portable, field-capable devices. LAMP primers were designed and validated to amplify a gene region unique to F. circinatum. Using a globally representative collection of F. circinatum isolates and other closely related species, we have demonstrated that the assay can be used to identify F. circinatum across its genetic diversity and that it is sensitive to as few as 10 cells from purified DNA extracts. The assay can also be used with a simple, pipette-free DNA extraction method and is compatible with testing symptomatic pine tissues in the field. This assay has the potential to facilitate diagnostic and surveillance efforts both in the laboratory and in the field and, thus, to reduce the spread and impact of pitch canker worldwide.

Condition of pine and larch forests adjacent to the reservoir within the Sayano-Shushensky Nature Reserve

Forests cover 60% of the Sayano-Shushensky Nature Reserve, 48% of which are larch and pine stands. The purpose of the research was to assess condition of Larix sibirica Ledeb. and Pinus sylvestris L. forests adjacent to the reservoir within the reserve. We conducted forest pathological inspection (for each species on four research plots), an integral assessment of the stands condition, macroscopic diagnostics and disease manifestations indicators estimation and studied model trees affected by xylophagous insects. Studied stands ranged from sustainable to ones with disturbed sustainability and their health status ranged from healthy to varying degrees of weakening. One of the main factors weakening forest stands were ground fires, leading to increased mortality of small-diameter trees that are less resistant to heat stress (most typical of P. sylvestris). Medium and large diameter trees post-fire mortality in L. sibirica forest stands was due to the Zeiraphera griseana feeding, and in relatively flat coastal areas due to repeating flooding. The presence of groups of trees dying from the top down were observed in pine forests growing on thin soils most likely due to an imbalance between the amount of required and available soil moisture. Diseases were not common and did not have a significant impact on the forest stands. Potentially harmful are the following diseases: in larch forests - European larch canker (pathogen - Lachnellula willkommii), in pine forests - Scots pine blister rust (pathogen - Cronartium pini). Xylophagous insects (dominated by Ips acuminatus) had an increased abundance and fed on dying trees in pine forests weakened after fires.

Phenotypic and Genomic Difference among Four Botryosphaeria Pathogens in Chinese Hickory Trunk Canker.

Botryosphaeria species are amongst the most widespread and important canker and dieback pathogens of trees worldwide, with B. dothidea as one of the most common Botryosphaeria species. However, the information related to the widespread incidence and aggressiveness of B. dothidea among various Botryosphaeria species causing trunk cankers is still poorly investigated. In this study, the metabolic phenotypic diversity and genomic differences of four Chinese hickory canker-related Botryosphaeria pathogens, including B. dothidea, B. qingyuanensis, B. fabicerciana, and B. corticis, were systematically studied to address the competitive fitness of B. dothidea. Large-scale screening of physiologic traits using a phenotypic MicroArray/OmniLog system (PMs) found B. dothidea has a broader spectrum of nitrogen source and greater tolerance toward osmotic pressure (sodium benzoate) and alkali stress among Botryosphaeria species. Moreover, the annotation of B. dothidea species-specific genomic information via a comparative genomics analysis found 143 B. dothidea species-specific genes that not only provides crucial cues in the prediction of B. dothidea species-specific function but also give a basis for the development of a B. dothidea molecular identification method. A species-specific primer set Bd_11F/Bd_11R has been designed based on the sequence of B. dothidea species-specific gene jg11 for the accurate identification of B. dothidea in disease diagnoses. Overall, this study deepens the understanding in the widespread incidence and aggressiveness of B. dothidea among various Botryosphaeria species, providing valuable clues to assist in trunk cankers management.

First report of Querciphoma minuta causing branch and stem canker in Platanus × hispanica in Germany

The London plane tree (Platanus × hispanica) is a long-lived drought tolerant tree, able to adapt to urban conditions and tolerate pollution, which is often grown as a park and avenue tree in Central Europe. In July and August 2022, branches from 20-year-old plane trees with large, dark-brown to red- brown lesions (Figure 1) extending from the bark (Figure 2a-b) to the heartwood (Figure 2c-d) were observed along an avenue in Cottbus, Germany. More than 100 trees (about 60% of the total) were diseased. Trees on the eastern side of the road, which were more exposed to sunlight, were significantly more affected than those on the west side, where sunlight exposure was reduced by larger trees in an adjacent park. Infected branches of different sizes (diameter 3–10 cm) were collected from four individual plane trees, with five samples being taken from each tree. From each branch, nine diseased samples were taken from the sapwood and heartwood, surface-sterilised and incubated on synthetic nutrient-poor agar (Sifin Diagnostics GmbH, Germany) for six days at 24 ±2°C under daylight conditions. Based on morphological characteristics, described in Crous and Groenewald (2017), Querciphoma minuta (syn. Q. carteri), a species within the Camarosporium complex was identified from all the samples. Conidiomata were medium brown, pycnidia, globose to eustromatic with 1–3 papillate ostioles, 150–250μm diameter (Figure 3a), with verruculose, septate setae with obtuse ends (Figure 3b-d). Conidia were solitary, hyaline, smooth, broadly ellipsoidal, aseptate and 4–6 × 3,5 μm in size. To confirm the identification, genomic DNA was extracted from four isolates and the internal transcribed spacer (ITS) region of rDNA was amplified using the ITS1/ITS4 primers (White et al., 1990). All consensus sequences (GenBank Accession Nos. OQ186736, OQ186741, OQ190053 and OQ190054) were identical. BLAST searches revealed 100 % sequence identity to Querciphoma minuta (KX359604.1, KF251209.1 and MN833930.1). A pathogenicity test was performed using two isolates (22-784B, 22–1121) on twenty healthy plane tree seedlings (Platanus × hispanica), 120 to 150 cm in height. Wounded stems were inoculated with mycelial plugs taken from the margin of a seven-day colony growing on potato dextrose agar (PDA) (sterile PDA agar plugs were used as controls) and sealed with plastic film. After six weeks of incubation at 24°C ±2°C and 75% humidity in the greenhouse, necrosis and vascular discolouration appeared on almost 98% of the sites inoculated with mycelium (Figures 4a-b and 5a-c), but not on the sites inoculated with sterile agar plugs (Figures 4c-d and Figure 5d). Necrosis extended mainly longitudinally from the inoculation point, causing brown to dark brown discolouration of the wood (Figure 3j). Lesions varied considerably, ranging from 1 to 5.3 cm in length for both isolates. Fungi were reisolated from all stem lesions and identified as above. Querciphoma minuta was confirmed in 85% of the inoculated sites, but in none of the controls, thus fulfilling Koch's postulates. Querciphoma minuta was until now known only to occur in oak, causing dieback (Carter 1941), or associated with veteranisation wounds in living oak trunks (Menkis et al., 2022). It is also pathogenic when associated with Coraebus florentinus in declining oak forests (Pinna et al., 2019). In 2022, Q. minuta was also found in Quercus petraea displaying dieback and large stem lesions in Spremberg, Germany (LELF, unpublished). Nevertheless, much remains to be discovered about the ecology of this fungus. This is the first report of Q. minuta as a potential canker pathogen of plane, suggesting that the pathogen has a larger host range of and a greater impact as a plant pathogen than previously suspected. T. Pfannenstill, M. Schemmel and R. Wichmann (LELF, P4, Plant Health Control) are gratefully acknowledged for sampling assistance. We thank C. Tielesch (LELF) and T. Wille (JKI) for laboratory assistance and T. Gaskin for language editing.

Identification and Characterization of Fungal Pathogens Causing Trunk and Branch Cankers of Almond Trees in Morocco

Canker symptoms were observed on almond trees in the Fez-Meknes region, Morocco. Isolations were conducted from the infected branch and trunk showing internal and external brown lesions. Four representative fungal isolates were screened, and their identities were confirmed by cultural traits and sequence analysis of DNA using two genes, the ITS region (internal transcribed spacer) and calmodulin (cmdA). The identified fungi were Curvularia hawaiiensis, Fusarium ambrosium, Lasiodiplodia theobromae, and Chondrestereum purpureum. The pathogenicity test on almond twigs revealed that these species were pathogenic to their host with different degrees of virulence, with Lasiodiplodia theobromae as the most virulent causing the longest necrotic lesion (285.17 mm) and the death of twigs. Physiological traits analysis of the above-mentioned fungi showed that the optimum mycelium growth response at different temperatures varies from 10 to 35 °C, while the pH ranges between 3.0 and 8.0. This study confirms the presence of canker pathogens on almond trees, which will contribute valuable information to improve the understanding of the contemporary status of almond trees, thus helping the improvement of the management of almond orchards. To our knowledge, all these fungi represent new records in Morocco and some of them are confirmed on the almond trees for the first time in the world.