Canker Research Articles

Comparative genomic analyses provide insight into the pathogenicity of three Pseudomonas syringae pv. actinidiae strains from Anhui Province, China

BackgroundPseudomonas syringae pv. actinidiae (Psa) is an important bacterial plant pathogen that causes severe damage to the kiwifruit industry worldwide. Three Psa strains were recently obtained from different kiwifruit orchards in Anhui Province, China. The present study mainly focused on the variations in virulence and genome characteristics of these strains based on the pathogenicity assays and comparative genomic analyses.ResultsThree strains were identified as biovar 3 (Psa3), along with strain QSY6 showing higher virulence than JZY2 and YXH1 in pathogenicity assays. The whole genome assembly revealed that each of the three strains had a circular chromosome and a complete plasmid. The chromosome sizes ranged from 6.5 to 6.6 Mb with a GC content of approximately 58.39 to 58.46%, and a predicted number of protein-coding sequences ranging from 5,884 to 6,019. The three strains clustered tightly with 8 Psa3 reference strains in terms of average nucleotide identity (ANI), whole-genome-based phylogenetic analysis, and pangenome analysis, while they were evolutionarily distinct from other biovars (Psa1 and Psa5). Variations were observed in the repertoire of effectors of the type III secretion system among all 15 strains. Moreover, synteny analysis of the three sequenced strains revealed eight genomic regions containing 308 genes exclusively present in the highly virulent strain QSY6. Further investigation of these genes showed that 16 virulence-related genes highlight several key factors, such as effector delivery systems (type III secretion systems) and adherence (type IV pilus), which might be crucial for the virulence of QSY6.ConclusionThree Psa strains were identified and showed variant virulence in kiwifruit plant. Complete genome sequences and comparative genomic analyses further provided a theoretical basis for the potential pathogenic factors responsible for kiwifruit bacterial canker.

Staphylococcus warneri, an unconventional plant pathogen involved in canker disease of almond and other Prunus species

AbstractBacterial canker disease of stone fruits is a major concern in stone fruit‐growing countries worldwide. Pseudomonas spp. and Xanthomonas arboricola pv. pruni (Xap) are the primary pathogens involved in this disease. In spring, summer and autumn 2016, symptoms like those produced by Pseudomonas spp. and Xap were observed in almond, apricot, peach and nectarine orchards in central provinces of Iran (Qom, Isfahan, and Chaharmahal and Bakhtiari). Gram‐positive cocci bacterial isolates were obtained from symptomatic trees. Following hypersensitivity and pathogenicity tests, isolates were divided into pathogenic and nonpathogenic groups, demonstrating the pathogenicity of some isolates on saplings of almond, peach and apricot. Multilocus sequence analysis was performed using the partial sequence of 16S rRNA region and four housekeeping genes, namely tuf, gap, dnaJ and rpoB, to determine the taxonomic classification of isolates, and revealed that pathogenic isolates identified as Staphylococcus warneri, while nonpathogenic isolates identified as S. warneri, S. epidermidis, S. hominis and S. saprophyticus. The isolates were further characterized by phenotypic and biochemical tests as well as by antibiotics assays. The unusual nature of the identified microorganism in the present study lies in the fact that, unlike most plant‐pathogenic agents, S. warneri is recognized worldwide as a cause of bacterial infections in humans and animals. Taken together, the bacterial canker disease caused by S. warneri appears to be a newly emerging disease of apricot, peach and almond trees.

Phytophthora and Phytopythium species associated with walnut dieback in plantations of northern Patagonia, Argentina

AbstractThe walnut industry in Argentina has grown in recent years, especially in northern Patagonia. However, this expansion has also brought new challenges, including the emergence of diseases threatening the health and productivity of walnut plantations. One of the most important diseases affecting walnut crops worldwide is ‘ink disease’ or ‘walnut dieback'. The aim of this study was to characterize ink disease, identify the aetiological agents, and estimate its incidence in walnut orchards of northern Patagonia, Argentina. Symptomatic trees were observed in the seven surveyed walnut orchards, showing a range of symptoms, including chlorosis and wilting, stem cankers and desiccation. In vivo assay and molecular analyses revealed that not only Phytophthora (P.) but also Phytopythium (Ph.) species are the causal agents of the disease, with P. drechsleri, Ph. mercuriale and Ph. vexans being the most virulent over the commonly used rootstock Juglans hindsii. Notably, Ph. vexans was the most frequently isolated pathogen, suggesting its prevalence and potential impact on the regional walnut industry. Overall, this study provides critical insights into the prevalence and impact of Phytophthora and Phytopythium species on walnut health in northern Patagonia. The presence of multiple pathogenic oomycete species raises concerns about the health of walnut orchards, as well as the potential risk of pathogen transmission to other fruit crops in the region. The findings will help growers to develop better cultural practices and implement measures to guarantee the sustainability of the walnut industry in Patagonia.

Phosphate solubilizing Pseudomonas and Bacillus combined with rock phosphates promoting tomato growth and reducing bacterial canker disease.

Nowadays, sustainable agriculture approaches are based on the use of biofertilizers and biopesticides. Tomato (Solanum lycopersicum L.) rhizosphere could provide rhizobacteria with biofertilizing and biopesticide properties. In this study, bacteria from the rhizosphere of tomato were evaluated in vitro for plant growth promotion (PGP) properties. Five Pseudomonas isolates (PsT-04c, PsT-94s, PsT-116, PsT-124, and PsT-130) and one Bacillus isolate (BaT-68s), with the highest ability to solubilize tricalcium phosphate (TCP) were selected for further molecular identification and characterization. Isolates showed phosphate solubilization up to 195.42 μg mL-1. All isolates showed phosphate solubilization by organic acid production. The six isolates improved seed germination and showed effective root colonization when tomato seeds were coated with isolates at 106 cfu g-1 in axenic soil conditions. Furthermore, the selected isolates were tested for beneficial effects on tomato growth and nutrient status in greenhouse experiments with natural rock phosphate (RP). The results showed that inoculated tomato plants in the presence of RP have a higher shoot and root lengths and weights compared with the control. After 60 days, significant increases in plant Ca, Na, P, protein, and sugar contents were also observed in inoculated seedlings. In addition, inoculated tomato seedlings showed an increase in foliar chlorophyll a and b and total chlorophyll, while no significant changes were observed in chlorophyll fluorescence. In greenhouse, two Pseudomonas isolates, PsT-04c and PsT-130, showed ability to trigger induced systemic resistance in inoculated tomato seedlings when subsequently challenged by Clavibacter michiganensis subsp. michiganensis, the causal agent of tomato bacterial canker. High protection rate (75%) was concomitant to an increase in the resistance indicators: total soluble phenolic compounds, phenylalanine-ammonia lyase, and H2O2. The results strongly demonstrated the effectiveness of phosphate-solubilizing bacteria adapted to rhizosphere as biofertilizers for tomato crops and biopesticides by inducing systemic resistance to the causal agent of tomato bacterial canker disease.

A preliminary biocontrol study: Isolation, characterization and genomic analysis of lytic phage TE against Pseudomonas syringae pv. syringae

AbstractTraditionally, apricot trees suffering from bacterial canker disease caused by Pseudomonas syringae pv. syringae were treated in orchards through the application of antibiotics or copper‐based drugs. However, considering the disadvantages of the long‐term use of chemical pesticides and antibiotics, there has been an increasing interest in phages, which have a lytic effect on bacteria, as biopesticides in recent years. In this study, we conducted a comprehensive examination of phage TE, isolated from wastewater in Turkey, from this perspective. Through scanning electron microscopy and phylogenetic analysis, we classified phage TE as belonging to the order Caudovirales and the family Strabovoridae. The phage TE exhibited a robust host lysis efficiency, with 280 infectious phage particles produced per bacterial cell in just 15 min. In experimental trials, treatment with phage TE resulted in an impressive 82% inhibition of bacterial canker disease in apricot leaves, highlighting its effectiveness as a phage therapy agent. Furthermore, phage TE exhibited long‐term stability when stored at 4°C without cryoprotectant and displayed tolerance to a wide temperature (−20°C to 50°C) and pH (2–12) range but its tolerance to UV‐C was very low. The genome and bioinformatics analyses of P. syringae phage TE have identified distinct protein level differences between other phages of the same species, despite sharing similarities at the nucleotide level. These findings suggest the potential use of phage TE as a biocontrol agent, offering a promising solution to control apricot bacterial canker.

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 more than 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.

Genome-wide identification and characterization of the sweet orange (Citrus sinensis) basic helix-loop-helix (bHLH) family reveals a role for CsbHLH085 as a regulator of citrus bacterial canker resistance

Citrus bacterial canker (CBC) is a harmful bacterial disease caused by Xanthomonas citri subsp. citri (Xcc), negatively impacting citrus production worldwide. The basic helix-loop-helix (bHLH) transcription factor family plays crucial roles in plant development and stress responses. This study aimed to identify and annotate bHLH proteins encoded in the Citrus sinensis genome and explore their involvement and functional importance in regulating CBC resistance. A total of 135 putative CsbHLHs TFs were identified and categorized into 16 subfamilies. Their chromosomal locations, collinearity, and phylogenetic relationships were comprehensively analyzed. Upon Xcc strain YN1 infection, certain CsbHLHs were differentially regulated in CBC-resistant and CBC-sensitive citrus varieties. Among these, CsbHLH085 was selected for further functional characterization. CsbHLH085 was upregulated in the CBC-resistant citrus variety, was localized in the nucleus, and had a transcriptional activation activity. CsbHLH085 overexpression in Citrus significantly enhanced CBC resistance, accompanied by increased levels of salicylic acid (SA), jasmonic acid (JA), reactive oxygen species (ROS), and decreased levels of abscisic acid (ABA) and antioxidant enzymes. Conversely, CsbHLH085 virus-induced gene silencing resulted in opposite phenotypic and biochemical responses. CsbHLH085 silencing also affected the expression of phytohormone biosynthesis and signaling genes involved in SA, JA, and ABA signaling. These findings highlight the crucial role of CsbHLH085 in regulating CBC resistance, suggesting its potential as a target for biotechnological-assisted breeding citrus varieties with improved resistance against phytopathogens.

Evaluation of cold plasma activated water composite reagent on kiwifruit plants with bacterial canker and the external qualities of kiwifruit after spray treatment

This study aimed to investigate the efficacy of cold plasma activated water (PAW) and its complex solution (PKC) in conjunction with kasugamycin copper oxychloride wettable powder (KC) against kiwifruit bacterial canker (KBC) caused by Pseudomonas syringae pv. actinidiae (PSA). Through morphological identification and 16S rDNA sequence analysis, Pantoea agglomerans was identified as the pathogen responsible for KBC in the 'Bairui' orchard. Results indicated that PKC exhibited the highest effectiveness in controlling KBC, particularly on mildly (II) infested plants, with a remarkable reduction of 63.64 % in PKC II disease index and a control efficiency of 38.64 %. Additionally, the application of PKC significantly increased fruit yield, improved fruit weight, and enhanced fruit morphology, resulting in a more uniform and refined fruit shape. Analysis of fruit skin conformational parameters revealed that KBC-affected fruit exhibited reduced hardness and brittleness, increased toughness, and thinner skin. These findings are pivotal for developing effective strategies to further prevent and manage KBC outbreaks. In future studies, further investigations could focus on elucidating the mechanism of action of PAW in complex solution with copper-based pesticides and the potential mechanism of inactivation of PSA.

Outbreak of Septoria leaf spot caused by Sphaerulina musiva on Populus × euramericana in China.

In June 2023, severe leaf spots were noted in Populus × euramericana cv 'Nanlin95' plantations located in the Nanjing Baguazhou Wetland Park (32°09'16.97″N, 118°48'16.74″E) of Jiangsu Province and Populus × canadensis cv 'Sacrau 79' and Populus × canadensis cv 'Guariento' in the Liyuan Village in Nanyang City (32°53'43.70″N, 112°17'29.12″E) of Henan Province, respectively. The disease incidence in both locations could reach 97.9% (556 out of 568 trees) and 98.9% (2409 out of 2436 trees), respectively. The initial symptoms appear as numerous small and circular spots (1.59 to 3.18 mm in diameter) with gray or tan centers and dark-brown margins on the leaves. As the spots age, they sometimes enlarge, often coalesce, and may extend down the petioles. Diseased leaves and petioles were both surface sterilized with 75% ethanol for 30 seconds. With the aid of a hand lens, pycnidia (brown to black, spherical in profile, 90 to 250 µm diam) were easily picked out in the center of the spots and subsequently transferred into 1 mL sterilized water for preparing the spore suspension plated on KV8 medium amended with 100 mg/liter streptomycin sulfate and 50 mg/liter chloramphenicol. After 12 days of incubation, 86 single-spore isolates were obtained and identified as typical Septoria-like fungi according to morphological features, including slow-growing, gray or black colonies with pink mucilaginous matrix and hyaline, straight or curved conidia (size = 25 to 59 × 3.5 to 4 μm; septa = 1 to 6). Species identification was further validated by PCR amplification and sequencing of the internal transcribed spacer (ITS) region with ITS1/ITS4 primer pairs. Multiple sequence alignments with ClustalW revealed that the obtained ITS sequences of 86 isolates were 100% identical to each other. A BLAST search in GenBank indicated that the selfsame sequences of two representative isolates (isolate BGZ11 of Jiangsu Province, accession no. OR660379; isolate KZB22 of Henan Province, accession no. OR711499) shared 99.8% identity (494 of 495 bp) and 100% identity (504 of 504 bp) with related sequences of Sphaerulina musiva (Peck) Quaedvlieg, Verkley, and Crous (syn. = Septoria musiva Peck) in GenBank (MN275187; KF251619), respectively. Furthermore, we used a S. musiva-specific PCR assay (Abraham et al. 2018) on symptomatic leaf samples collected from the plantation. Each sample consisted of 20 cut-out leaf spots per leaf. Eight of the 10 samples were positive for S. musiva DNA. To confirm pathogenicity, six sterile tissue culture of poplar plants (Populus trichocarpa and Populus × euramericana cv 'Nanlin895') were respectively transplanted into pots and grown in a greenhouse for a week and for a month with an 18-h photoperiod augmented with sodium lamps and a 20°C (day)/16°C (night) temperature regime. Inoculations were conducted by spraying the plants with conidia suspension (106 conidia/mL) (LeBoldus et al. 2010). Control plants were sprayed with distilled water. Leaf spots were developed on the inoculated P. trichocarpa leaves at one week and P. × euramericana cv 'Nanlin895' leaves at 10 days after inoculation while no symptoms were observed on the control plants. The fungus S. musiva was successfully reisolated from all symptomatic leaves fulfilling Koch's postulates. Sphaerulina musiva only causes an endemic leaf spot disease on its natural North American host Populus. deltoides (Feau et al. 2010; Ostry 1987). However, on susceptible Populus species (e.g., P. balsamifera, P. trichocarpa, P. maximowiczii) and hybrids, S. musiva causes not only leaf spots but also severely damaging stem and branch cankers (Jeger et al. 2018; LeBoldus et al. 2009; Sondreli et al. 2020). To our knowledge, this is the first report of S. musiva causing leaf spots on poplar in China. Large-scale timber imports (e.g., cut branches, isolated bark, wood with and without bark) potentially lead to anthropogenic-facilitated transport of this pathogen. This outbreak of Septoria leaf spot underscores the potential threat of this pathogen to P. × euramericana in China, where it is widely planted as a keystone forestry species.

Phylogenomic analyses and comparative genomics of Pseudomonas syringae associated with almond (Prunus dulcis) in California.

We sequenced and comprehensively analysed the genomic architecture of 98 fluorescent pseudomonads isolated from different symptomatic and asymptomatic tissues of almond and a few other Prunus spp. Phylogenomic analyses, genome mining, field pathogenicity tests, and in vitro ice nucleation and antibiotic sensitivity tests were integrated to improve knowledge of the biology and management of bacterial blast and bacterial canker of almond. We identified Pseudomonas syringae pv. syringae, P. cerasi, and P. viridiflava as almond canker pathogens. P. syringae pv. syringae caused both canker and foliar (blast) symptoms. In contrast, P. cerasi and P. viridiflava only caused cankers, and P. viridiflava appeared to be a weak pathogen of almond. Isolates belonging to P. syringae pv. syringae were the most frequently isolated among the pathogenic species/pathovars, composing 75% of all pathogenic isolates. P. cerasi and P. viridiflava isolates composed 8.3 and 16.7% of the pathogenic isolates, respectively. Laboratory leaf infiltration bioassays produced results distinct from experiments in the field with both P. cerasi and P. syringae pv. syringae, causing significant necrosis and browning of detached leaves, whereas P. viridiflava conferred moderate effects. Genome mining revealed the absence of key epiphytic fitness-related genes in P. cerasi and P. viridiflava genomic sequences, which could explain the contrasting field and laboratory bioassay results. P. syringae pv. syringae and P. cerasi isolates harboured the ice nucleation protein, which correlated with the ice nucleation phenotype. Results of sensitivity tests to copper and kasugamycin showed a strong linkage to putative resistance genes. Isolates harbouring the ctpV gene showed resistance to copper up to 600 μg/ml. In contrast, isolates without the ctpV gene could not grow on nutrient agar amended with 200 μg/ml copper, suggesting ctpV can be used to phenotype copper resistance. All isolates were sensitive to kasugamycin at the label-recommended rate of 100μg/ml.

Unveiling kiwifruit TCP genes: evolution, functions, and expression insights

ABSTRACT The TEOSINTE-BRANCHED1/CYCLOIDEA/PROLEFERATING-CELL-FACTORS (TCP) gene family is a plant-specific transcriptional factor family involved in leaf morphogenesis and senescence, lateral branching, hormone crosstalk, and stress responses. To date, a systematic study on the identification and characterization of the TCP gene family in kiwifruit has not been reported. Additionally, the function of kiwifruit TCPs in regulating kiwifruit responses to the ethylene treatment and bacterial canker disease pathogen (Pseudomonas syringae pv. actinidiae, Psa) has not been investigated. Here, we identified 40 and 26 TCP genes in Actinidia chinensis (Ac) and A. eriantha (Ae) genomes, respectively. The synteny analysis of AcTCPs illustrated that whole-genome duplication accounted for the expansion of the TCP family in Ac. Phylogenetic, conserved domain, and selection pressure analysis indicated that TCP family genes in Ac and Ae had undergone different evolutionary patterns after whole-genome duplication (WGD) events, causing differences in TCP gene number and distribution. Our results also suggested that protein structure and cis-element architecture in promoter regions of TCP genes have driven the function divergence of duplicated gene pairs. Three and four AcTCP genes significantly affected kiwifruit responses to the ethylene treatment and Psa invasion, respectively. Our results provided insight into general characters, evolutionary patterns, and functional diversity of kiwifruit TCPs.

Genome-wide analysis and expression profiling of the HD-ZIP gene family in kiwifruit

The homeodomain-leucine zipper (HD-Zip) gene family plays a pivotal role in plant development and stress responses. Nevertheless, a comprehensive characterization of the HD-Zip gene family in kiwifruit has been lacking. In this study, we have systematically identified 70 HD-Zip genes in the Actinidia chinensis (Ac) genome and 55 in the Actinidia eriantha (Ae) genome. These genes have been categorized into four subfamilies (HD-Zip I, II, III, and IV) through rigorous phylogenetic analysis. Analysis of synteny patterns and selection pressures has provided insights into how whole-genome duplication (WGD) or segmental may have contributed to the divergence in gene numbers between these two kiwifruit species, with duplicated gene pairs undergoing purifying selection. Furthermore, our study has unveiled tissue-specific expression patterns among kiwifruit HD-Zip genes, with some genes identified as key regulators of kiwifruit responses to bacterial canker disease and postharvest processes. These findings not only offer valuable insights into the evolutionary and functional characteristics of kiwifruit HD-Zips but also shed light on their potential roles in plant growth and development.

Chrysoporthe brasiliensis sp. nov. pathogenic to Melastomataceae in southeast Brazil

Species in the Melastomataceae (Myrtales) include trees and woody shrubs that are amongst the most common hosts of Chrysoporthe and related fungi. These fungi cause stem cankers, branch death and in extreme cases, kill their hosts. Chrysoporthe-like fungi were observed on Miconia spp. and Rhynchanthera grandiflora (Melastomataceae) plants during tree disease surveys in south-eastern Brazil including the states of Minas Gerais and Rio de Janeiro. The aims of this study were to isolate and identify the fungi utilising morphological characteristics and phylogenetic analyses. This led to the identification of a new species of Chrysoporthe described here as Chrysoporthe brasilensis sp.nov. Inoculations were conducted on R. grandiflora and M. theaezans, showing that C. brasiliensis is an aggressive pathogen. This study adds to a growing number of reports of new and pathogenic species of Chrysoporthe that potentially threaten native Myrtales globally, including important trees such as Eucalyptus, both in natural ecosystems and in planted forests.

Physicochemical Characterization of Novel Bacteriophages of Pseudomonas syringe from Northwest Iran.

Bacterial canker, caused by Pseudomonas syringae, is a devastating disease of stone fruit trees worldwide. The bacterium has a broad host range and a high capacity for adaptation and dissemination, owing to its high mutation rate and horizontal gene transfer. Traditional control methods based on copper compounds and antibiotics have resulted in the development of resistance in the bacterial population. Thus, alternative approaches are needed, such as phage therapy. This study aimed to characterize the physicochemical and biological properties of novel Pseudomonas syringae pv. syringae (Pss)-specific phages isolated from the soils of northwestern Iran. Seventy-five phage isolates were obtained, and their host range was determined against various bacterial pathogens. Five phages exhibiting the highest lytic activity against Pss and a narrow host range were selected for subsequent analysis. The stability of the selected phages was assessed under different conditions such as ultraviolet irradiation, temperature, pH, NaCl concentration, and chloroform exposure. The selected phages demonstrated significant effectiveness invivo, exerting substantial suppression on the population of Pss. This reduction was observed for both individual phages and when the phages were utilized as a mixture. The findings indicate that phages have the potential to be used as biocontrol agents in agriculture.

Eradication measures and products that inhibit in vitro mycelial growth of the fungus Neoscytalidium dimidiatum, the etiological agent of pitaya canker

The objective of this work was to identify the causal agent of the circular necrotic lesion present in cladodes and pitaya fruits, to test in vitro different products with potential fungicidal effect and to evaluate the effect of different eradicating products on infected pruned cladodes. Fruits and cladodes with symptoms of pitaya canker were analyzed using a magnifying glass and microscope. Isolation in PDA and inoculation was also carried out in healthy cladodes to observe symptoms. The isolated fungus was placed to grow in PDA culture medium previously mixed with the products; Castor oil, Prevam, Biometal copper, Kellus immune, Primecur, Kaumin, Bombardeiro, Serenade, Bordamil, Dithane, Bravonil, Mertin, Rovral, Sumilex, Mythos, Cantus, Fluazinam Nortox, Comet, Cercobin, Forum, Socre Flex, Cabrio Top, Orkestra, Opera, Nativo, Fusion, Tridium, Priori Xtra and Evolution. The products were applied to infected cladodes; Quaternary ammonia, Fortalece, Bordamil, Calda sulfocaucica all in concentrations of 5% and control with the objective of observing sporulation, spore germination and decomposition time. Pitaya cladodes and fruits presented stem canker caused by Neoscytalidium dimidiatum. The products; Prevam, Bordamil, Dithane, Mertin, Rovral, Sumilex, Fluazinam Nortox, Comet, Socre Flex, Cabrio Top, Orkestra, Opera, Nativo, Fusion, Tridium, Priori Xtra and Evolution at the doses tested completely inhibited the mycelial growth of the N. dimidiatum fungus. The products; Bordamil®, Sulfocaucic solution, Quaternary Ammonia and Fortalece® in concentrations of 5% accelerated the decomposition of the treated cladode and reduced the formation and germination of N. dimidiatum conidia.

Morphology, phylogeny and pathogenicity of fungal species associated with leaf blight and stem canker of Theobroma cacao in Malaysia

AbstractMalaysia stands prominently among Asia's key cocoa‐producing countries. In the cocoa season of 2022–2023, Malaysia demonstrated its contribution to the industry, with an estimated production of around 364,000 tonnes of cacao bean grindings. Nonetheless, fungal diseases pose undeniable challenges to the cocoa sector. Extensive sampling conducted between September 2018 and March 2019 across multiple states in Malaysia revealed concerning symptoms of leaf blight and stem canker affecting Theobroma cacao plants. The aim of this study was to identify and characterize fungal species associated with leaf blight and stem canker of T. cacao in Malaysia through morphological, molecular and pathogenicity analyses. Morphological and molecular phylogenetic analyses using multiple DNA regions (rDNA internal transcribed spacer [ITS], TEF1 and TUB2) were performed and identified 40 fungal isolates found in this study as Diaporthe tulliensis (17 isolates), Fusarium solani (seven isolates), Fusarium proliferatum (six isolates) and Neopestalotiopsis clavispora (10 isolates). Pathogenicity tests with mycelial plugs and wound treatments showed that D. tulliensis and N. clavispora were responsible for causing leaf blight whereas D. tulliensis, F. solani and F. proliferatum caused stem canker of T. cacao. The present study provides insights into disease aetiology and symptomatology that may be useful in planning effective disease management for the host plant.

Evolution of decreased sensitivity to azole fungicides in western European populations of Plenodomus lingam (Phoma stem canker on oilseed rape)

AbstractPlenodomus lingam (Leptosphaeria maculans) and P. biglobosus (L. biglobosa) are fungi causing Phoma leaf spot/stem canker, an international damaging disease of oilseed rape (Brassica napus) and other brassicas. In Europe, fungicides used for disease management are mainly sterol 14α‐demethylase (CYP51) inhibitors (DMIs/azoles); quinone‐outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs) are also used. Decreased DMI sensitivity has emerged in Australian and eastern European P. lingam populations and is mediated by CYP51 promoter inserts resulting in target site overexpression. In this study using in vitro sensitivity testing, we report decreased DMI (prothioconazole‐desthio, mefentrifluconazole) sensitivity in modern western European P. lingam isolates (collected 2022–2023) compared to older baseline (1992–2005) isolates. Around 85% of modern western European P. lingam isolates collected, for which the CYP51 promoter region was sequenced, carried a promoter insert, but target site alterations were not detected. Six different CYP51 promoter inserts were identified, most commonly a 237 bp fragment of the Sahana transposable element. Inserts were associated with an approximately 3‐ to 10‐fold decrease in sensitivity to the DMIs tested. In contrast to P. lingam, PCR screening revealed CYP51 promoter inserts were absent in modern western European P. biglobosus isolates (2021–2023). Combined data indicate P. lingam isolates lacking an insert were similarly (or slightly more) sensitive to the DMIs tested for P. biglobosus, whereas those carrying an insert were slightly less sensitive than P. biglobosus. No evidence for substantive sensitivity shifts to the QoI (pyraclostrobin) or SDHI (boscalid) fungicides tested was obtained for either Plenodomus species.

Transient expression of anti-HrpE scFv antibody reduces the hypersensitive response in non-host plant against bacterial phytopathogen Xanthomonas citri subsp. citri

Citrus canker is a bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) that affects the citrus industry worldwide. Hrp pili subunits (HrpE), an essential component of Type III secretion system (T3SS) bacteria, play a crucial role in the pathogenesis of Xcc by transporting effector proteins into the host cell and causing canker symptoms. Therefore, development of antibodies that block HrpE can suppress disease progression. In this study, a specific scFv detecting HrpE was developed using phage display technique and characterized using sequencing, ELISA, Western blotting, and molecular docking. In addition, a plant expression vector of pCAMBIA-scFvH6 was constructed and agroinfiltrated into Nicotiana tabacum cv. Samson leaves. The hypersensitive response (HR) in the leaves of transformed and non-transformed plants was evaluated by inoculating leaves with Xcc. After three rounds of biopanning of the phage library, a specific human scFv antibody, named scFvH6, was identified that showed high binding activity against HrpE in ELISA and Western blotting. Molecular docking results showed that five intermolecular hydrogen bonds are involved in HrpE-scFvH6 interaction, confirming the specificity and high binding activity of scFvH6. Successful transient expression of pCAMBIA-scFvH6 in tobacco leaves was verified using immunoassay tests. The binding activity of plant-produced scFvH6 to detect HrpE in Western blotting and ELISA was similar to that of bacterial-produced scFvH6 antibody. Interestingly, tobacco plants expressing scFvH6 showed a remarkable reduction in HR induced by Xcc compared with control plants, so that incidence of necrotic lesions was significantly higher in non-transformed controls (≥ 1.5 lesions/cm2) than in the plants producing scFvH6 (≤ 0.5 lesions/cm2) after infiltration with Xcc inoculum. Our results revealed that the expression of scFvH6 in tobacco leaves can confer resistance to Xcc, indicating that this approach could be considered to provide resistance to citrus bacterial canker disease.

Genome-wide analysis of NPR1-like genes in citrus species and expression analysis in response to citrus canker (Xanthomonas axonopodis pv. citri).

Citrus fruits, revered for their nutritional value, face significant threats from diseases like citrus canker, particularly impacting global citrus cultivation, notably in Pakistan. This study delves into the critical role of NPR1-like genes, the true receptors for salicylic acid (SA), in the defense mechanisms of citrus against Xanthomonas axonopodis pv. citri (Xcc). By conducting a comprehensive genome-wide analysis and phylogenetic study, the evolutionary dynamics of Citrus limon genes across diverse citrus cultivars are elucidated. Structural predictions unveil conserved domains, such as the BTB domain and ankyrin repeat domains, crucial for the defense mechanism. Motif analysis reveals essential conserved patterns, while cis-regulatory elements indicate their involvement in transcription, growth, response to phytohormones, and stress. The predominantly cytoplasmic and nuclear localization of NPR1-like genes underscores their pivotal role in conferring resistance to various citrus species. Analysis of the Ks/Ka ratio indicates a purifying selection of NPR1-like genes, emphasizing their importance in different species. Synteny and chromosomal mapping provide insights into duplication events and orthologous links among citrus species. Notably, Xac infection stimulates the expression of NPR1-like genes, revealing their responsiveness to pathogenic challenges. Interestingly, qRT-PCR profiling post-Xac infection reveals cultivar-specific alterations in expression within susceptible and resistant citrus varieties. Beyond genetic factors, physiological parameters like peroxidase, total soluble protein, and secondary metabolites respond to SA-dependent PR genes, influencing plant characteristics. Examining the impact of defense genes (NPR1) and plant characteristics on disease resistance in citrus, this study marks the inaugural investigation into the correlation between NPR1-associated genes and various plant traits in both susceptible and resistant citrus varieties to citrus bacterial canker.

Botryosphaeria dothidea causes stem canker of Idesia polycarpa in China

AbstractIdesia polycarpa is a woody oil plant with great development and application prospects. However, stem canker disease of I. polycarpa was frequently observed in Henan and Hubei provinces of China in 2020–2022. The seriousness of the disease caused a large number of trees deaths, which affected seedling production and fruit yield, restricting the development of the I. polycarpa industry. We performed isolation, purification and pathogenicity analysis of canker samples. Pathogenicity tests reproduced typical canker disease symptoms on detached branches of I. polycarpa. Based on morphological observations, conidial morphology and phylogenetic analysis of isolates with high similarity to the rDNA internal transcribed spacer (ITS), EF‐1α and TUB2 sequences screened in GenBank by BLAST, the pathogen of canker disease on I. polycarpa was identified as Botryosphaeria dothidea. This is the first report of B. dothidea as the pathogen of stem canker of I. polycarpa.