Bottlebrush and Myrtle twig canker caused by Neopestalotiopsis species: an emerging canker-causing group of fungi in Italy.

Pestalotioid fungi were isolated in pure culture from symptomatic plants of Callistemonlaevis, C.viminalis, Lumaapiculata (marketed as "Myrtusluma"), Myrtuscommunissubsp.tarentina, and M.communisvar.microphylla (M.communis 'Microphylla'), showing twig canker, dieback and defoliation. The isolates were identified to species by ITS, tef1 and tub2 sequences, which revealed the presence of six species of Neopestalotiopsis (N.camelliae-oleiferae, N.hispanica, N.iberica, N.rosae, N.rosicola, and N.zakeelii) and one species of Pestalotiopsis (P.biciliata). While most species were isolated only once or twice, the majority of isolates belonged to N.rosae (13) and N.hispanica (8). Pathogenicity was investigated by pathogenicity tests on all hosts, which confirmed the pathogenicity of all Neopestalotiopsis species on at least some of the hosts tested, while P.biciliata did not cause any disease symptoms. Neopestalotiopsishispanica and N.rosae caused symptoms in all hosts of the present study, while the other Neopestalotiopsis species tested showed no symptoms on Lumaapiculata.

Management of Tomato Bacterial Canker Disease by the Green Fabricated Silver Nanoparticles.

Bacterial canker disease caused by Clavibacter michiganensis is a substantial threat to the cultivation of tomatoes, leading to considerable economic losses and global food insecurity. Infection is characterized by white raised lesions on leaves, stem, and fruits with yellow to tan patches between veins, and marginal necrosis. Several agrochemical substances have been reported in previous studies to manage this disease but these were not ecofriendly. Thus present study was designed to control the bacterial canker disease in tomato using green fabricated silver nanoparticles (AgNps). Nanosilver particles (AgNPs) were synthesized utilizing Moringa oleifera leaf extract as a reducing and stabilizing agent. Synthesized AgNPs were characterized using UV-visible spectroscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), energy-dispersive X-ray (EDX), and Fourier transform infrared spectrometry (FTIR). FTIR showed presence of bioactive compounds in green fabricated AgNPs and UV-visible spectroscopy confirmed the surface plasmon resonance (SPR) band in the range of 350 nm to 355 nm. SEM showed the rectangular segments fused together, and XRD confirmed the crystalline nature of the synthesized AgNPs. The presence of metallic silver ions was confirmed by an EDX detector. Different concentrations (10, 20, 30, and 40 ppm) of the green fabricated AgNPs were exogenously applied on tomato before applying an inoculum of Clavibacter michigensis to record the bacterial canker disease incidence at different day intervals. The optimal concentration of AgNPs was found to be 30 µg/mg that exhibited the most favorable impact on morphological (shoot length, root length, plant fresh and dry weights, root fresh and dry weights) and physiological parameters (chlorophyll contents, membrane stability index, and relative water content) as well as biochemical parameters (proline, total soluble sugar and catalase activity). These findings indicated a noteworthy reduction in biotic stress through the increase of both enzymatic and non-enzymatic activities by the green fabricated AgNPs. This study marks a first biocompatible approach in assessing the potential of green fabricated AgNPs in enhancing the well-being of tomato plants that affected with bacterial canker and establishing an effective management strategy against Clavibacter michiganensis. This is the first study suggests that low concentration of green fabricated nanosilvers (AgNPs) from leaf extract of Moringa oleifera against Clavibacter michiganensis is a promisingly efficient and eco-friendly alternative approach for management of bacterial canker disease in tomato crop.

Novel phages of Pseudomonas syringae unveil numerous potential auxiliary metabolic genes.

Relatively few phages that infect plant pathogens have been isolated and investigated. The Pseudomonas syringae species complex is present in various environments, including plants. It can cause major crop diseases, such as bacterial canker on apricot trees. This study presents a collection of 25 unique phage genomes that infect P. syringae. These phages were isolated from apricot orchards with bacterial canker symptoms after enrichment with 21 strains of P. syringae. This collection comprises mostly virulent phages, with only three being temperate. They belong to 14 genera, 11 of which are newly discovered, and 18 new species, revealing great genetic diversity within this collection. Novel DNA packaging systems have been identified bioinformatically in one of the new phage species, but experimental confirmation is required to define the precise mechanism. Additionally, many phage genomes contain numerous potential auxiliary metabolic genes with diversified putative functions. At least three phages encode genes involved in bacterial tellurite resistance, a toxic metalloid. This suggests that viruses could play a role in bacterial stress tolerance. This research emphasizes the significance of continuing the search for new phages in the agricultural ecosystem to unravel novel ecological diversity and new gene functions. This work contributes to the foundation for future fundamental and applied research on phages infecting phytopathogenic bacteria.

First Report of Pseudomonas amygdali pv. morsprunorum causing Bacterial Canker in Sweet Cherry Orchards in Washington State.

In 2023, an outbreak of bacterial canker disease (BCD) in sweet cherry orchards caused significant economic losses to growers and nurseries in the Pacific Northwest, USA (Fig. S1). The cherry industry in Washington State alone is valued at over $800 million (USDA NASS, 2022). BCD poses a recurring threat to the state's sweet cherry [Prunus avium (L.) L.] orchards, especially young and newly planted orchards. Three Pseudomonas species, including P. syringae pv. syringae (Pss), P. amygdali pv. morsprunorum (Pam) (formerly P. syringae pv. morsprunorum Race 1, Psm1), and P. avellanae pv. morsprunorum (formerly P. syringae pv. morsprunorum Race 2, Psm2), have been reported to be associated with BCD in sweet cherries (Hulin et al. 2019). While Pss is widely prevalent in the United States, Pam has only been reported in Michigan (Renick et al., 2008) as well as in Europe, Central America, South Africa and Australia (Hulin et al. 2019) . In 2023, we surveyed more than 60 cherry orchards and collected hundreds of canker samples from newly planted up to 8-year-old trees. BCD prevalence ranged from 40-100% in cherry orchards, leading to the removal of hundreds of thousands of trees. Affected cherry trees exhibited characteristic bacterial canker symptoms, including dead bud, canker, and gummosis (Fig. S1). Bacteria were isolated from canker tissues or ooze on King's B (KB) agar plates (King et al., 1954) and more than 300 fluorescent Pseudomonas isolates were obtained from 12 symptomatic sweet cherry cultivars. PCR results using Pss- and Pam- specific primers (SyrB and Psm1, Table S1) (Sorensen et al., 1998; Kaluzna et al., 2016) revealed that 91.9% and 8% isolates were tested positive for SyrB and Psm1, indicating that these isolates potentially belong to Pss and Pam, respectively. Pathogenicity tests using immature cherries cv. Sentina showed that all isolates caused typical necrotic lesions and could be re-isolated and re-identified as Pss and Pam, thus completing Koch's postulates. The identity of three Pam representative isolates (S79, S158, S202) was further confirmed by comparing gyrD and rpoD housekeeping genes as well as 16S rRNA gene sequence with other Pam strains in GenBank (Parkinson et al., 2010; Gomila et al., 2017). Blast searches against GenBank using gyrB (GenBank accession numbers PP357444-PP357446), rpoD (PP357447-PP357449) and 16S rRNA (GenBank accession numbers PP421223-PP421225) gene sequences, ranging from 520 to 859bp, matched those of the Pam isolates (GenBank accession numbers CP026558 or PP218075) with 100% homology and 100% query coverage, further indicating that these isolates are indeed Pam. This represents the first documented record of Pam causing BCD in the Pacific Northwest, USA, suggesting the complexity of the disease, which underscores the need for effective management strategies for cherry growers in the region.

Endophytic fungal isolates from apple tissue: Latent pathogens lurking within?

Fungal endophytes inhabit a similar ecological niche to that occupied by many phytopathogens, with several pathogens isolated from healthy tissues in their latent phase. This study aimed to evaluate the pathogenicity, the colonisation ability, and the enzyme activity of 37 endophytic fungal isolates recovered from apparently healthy apple shoot and leaf tissues. The pathogenicity of the isolates was assessed on 'Royal Gala' and 'Braeburn' fruit and detached 'Royal Gala' shoots. For the non-pathogenic isolates, their ability to endophytically colonise detached 'Royal Gala' shoots was evaluated. Enzyme activity assays were undertaken to determine whether the pathogenicity of the endophytes was related to the production of the extracellular enzymes, amylase, cellulase, pectinase, protease, and xylanase. Of the 37 isolates studied, eight isolates, representing the genera Colletotrichum, Diaporthe, Fusarium, and Penicillium, were shown to be pathogenic on both apple shoots and fruit. Two isolates identified as Trichoderma atroviride, were pathogenic only on shoots, and three isolates, representing the genus Diaporthe, were pathogenic only on fruit. Of the remaining 24 isolates, 22 (Biscogniauxia (n = 8), Chaetomium (n = 4), Trichoderma (n = 3), Epicoccum (n = 2), Neosetophoma (n = 2), Xylaria (n = 1), Daldinia (n = 1), and Paraphaeosphaeria (n = 1)) were recovered from the inoculated apple shoots but two failed to colonise the shoot tissues. Of the isolates tested, 20 produced amylase, 15 cellulase, 25 pectinase, 26 protease, and 13 xylanase. There was no correlation between the range and type of enzymes produced by the isolates and their pathogenicity or ability to endophytically colonise the shoot tissue. The study showed that approximately one-third (13/37) of the isolates recovered from the apparently healthy apple shoot tissues were observed as latent pathogens. The isolates that did not cause disease symptoms may have the ability to reduce colonisation of apple tissues by pathogens including Neonectria ditissima associated with European canker of apple.

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.

Two novel Raoultella species associated with bleeding cankers of broadleaf hosts, Raoultella scottia sp. nov. and Raoultella lignicola sp. nov.

Seventeen Gram-negative, facultatively anaerobic bacterial strains were isolated from bleeding cankers of various broadleaf hosts and oak rhizosphere soil in Great Britain. The strains were tentatively identified as belonging to the genus Raoultella based on 16S rRNA gene sequencing. Multilocus sequence analysis (MLSA), based on four protein-encoding genes (fusA, leuS, pyrG, and rpoB), separated the strains into three clusters within the Raoultella genus clade. The majority of strains clustered with the type strain of Raoultella terrigena, with the remaining strains divided into two clusters with no known type strain. Whole genome sequencing comparisons confirmed these two clusters of strains as belonging to two novel Raoultella species which can be differentiated phenotypically from their current closest phylogenetic relatives. Therefore, two novel species are proposed: Raoultella scottia sp. nov. (type strain = BAC 10a-01-01T = LMG 33072T = CCUG 77096T) and Raoultella lignicola sp. nov. (type strain = TW_WC1a.1T = LMG 33073T = CCUG 77094T).

Comparative mineral and biochemical characterization of Citrus reticulata fruits and leaves to citrus canker pathogens, Xanthomonas axonopodis.

Pakistan's economy greatly benefits from citrus production since these fruits are sold and consumed all over the world. Although citrus fruits are easy to cultivate, they are susceptible to diseases caused by bacteria, viruses, and fungi. These challenges, as well as difficulties in obtaining the proper nutrients, might negatively impact fruit yields and quality. Citrus canker is another complicated problem caused by the germ Xanthomonas axonopodis. This germ affects many types of citrus fruits all over the world. This study looked closely at how citrus canker affects the leaves and the quality of the fruit in places like Sargodha, Bhalwal, Kotmomin, and Silanwali, which are big areas for growing citrus in the Sargodha district. What we found was that plants without the disease had more chlorophyll in their leaves compared to the sick plants. Also, the healthy plants had better amounts of important minerals like calcium, magnesium, potassium, and phosphorus in their fruits. But the fruits with the disease had too much sodium, and the iron levels were a bit different. The fruits with the disease also didn't have as much of something that protects them called antioxidants, which made them more likely to get sick. This study helps us understand how citrus canker affects plants and fruit, so we can think of ways to deal with it.

Clarification of the infection pattern of Xanthomonas citri subsp. citri on citrus fruit by artificial inoculation.

BACKGROUND: Citrus canker is a significant bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) that severely impedes the healthy development of the citrus industry. Especially when citrus fruit is infected by Xcc, it will reduce or even lost its commercial value. However, due to the prolonged fruiting cycle and intricate structure, much less research progress had been made in canker disease on fruit than on leaf. In fact, limited understanding has been achieved on canker development and the response to Xcc infection in fruit. RESULTS: Herein, the progression of canker disease on sweet orange fruit was tracked in the field. Results indicated that typical lesions initially appear on the sepal, style residue, nectary disk, epicarp, and peduncle of young fruits after petal fall. The susceptibility of fruits to Xcc infection diminished as the fruit developed, with no new lesions forming at the ripening stage. The establishment of an efficient method for inoculating Xcc on fruit as well as the artificial inoculation throughout the fruit's developmental cycle clarified this infection pattern. Additionally, microscopic observations during the infection process revealed that Xcc invasion caused structural changes on the surface and cross-section of the fruit. CONCLUSIONS: An efficient system for inoculation on citrus fruit with Xcc was established, by which it can serve for the evaluation of citrus germplasm for canker disease resistance and systematic research on the interactions between Xcc and citrus fruits.

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.

A novel virulence gene, cviA1 of Clavibacter michiganensis for necrosis development in the Nicotiana benthamiana plant.

Clavibacter michiganensis is a Gram-positive bacterium that causes diverse disease symptoms in tomatoes and Nicotiana benthamiana, a surrogate host plant, including canker, blister lesions, and wilting. Previously, we reported that C. michiganensis also causes necrosis in N. benthamiana leaves. Here, to identify novel virulence genes of C. michiganensis required for necrosis development in N. benthamiana leaves, we screened 1,862 transposon-inserted mutants and identified a mutant strain that exhibited weak and delayed necrosis, whereas there was no discernible difference in blister lesions, canker, or wilting symptoms. Notably, this mutant caused canker similar to that of the wild-type strain, but caused mild wilting in tomato. This mutant carried a transposon in a chromosomal gene, called Clavibactervirulence gene A1 (cviA1). CviA1 encodes a 180-amino acid protein with a signal peptide (SP) at the N-terminus and two putative transmembrane domains (TMs) at the C-terminus. Interestingly, deletion of the SP or the C-terminus, including the two putative TMs, in CviA1 failed to restore full necrosis in the mutant, highlighting the importance of protein secretion and the putative TMs for necrosis. A paralog of cviA1, cviA2 is located on the large plasmid pCM2 of C. michiganensis. Despite its high similarity to cviA1, the introduction of cviA2 into the cviA1 mutant strain did not restore virulence. Similarly, the introduction of cviA1 into the Clavibacter capsici type strain PF008, which initially lacks cviA1, did not enhance necrosis symptoms. These results reveals that the chromosomal cviA1 gene in C. michiganensis plays an important role in necrosis development in N. benthamiana leaves.

Genome-wide identification of walnut (Juglans regia) PME gene family members and expression analysis during infection with Cryptosphaeria pullmanensis pathogens.

Walnuts exhibit a higher resistance to diseases, though they are not completely immune. This study focuses on the Pectin methylesterase (PME) gene family to investigate whether it is involved in disease resistance in walnuts. These 21 genes are distributed across 12 chromosomes, with four pairs demonstrating homology. Variations in conserved motifs and gene structures suggest diverse functions within the gene family. Phylogenetic and collinear gene pairs of the PME family indicate that the gene family has evolved in a relatively stable way. The cis-acting elements and gene ontology enrichment of these genes, underscores their potential role in bolstering walnuts' defense mechanisms. Transcriptomic analyses were conducted under conditions of Cryptosphaeria pullmanensis infestation and verified by RT-qPCR. The results showed that certain JrPME family genes were activated in response, leading to the hypothesis that some members may confer resistance to the disease.

GDP-mannose pyrophosphorylase is an efficient target in Xanthomonas citri for citrus canker control.

Xanthomonas citri subsp. citri (Xcc) is a bacterium that causes citrus canker, an economically important disease that results in premature fruit drop and reduced yield of fresh fruit. In this study, we demonstrated the involvement of XanB, an enzyme with phosphomannose isomerase (PMI) and guanosine diphosphate-mannose pyrophosphorylase (GMP) activities, in Xcc pathogenicity. Additionally, we found that XanB inhibitors protect the host against Xcc infection. Besides being deficient in motility, biofilm production, and ultraviolet resistance, the xanB deletion mutant was unable to cause disease, whereas xanB complementation restored wild-type phenotypes. XanB homology modeling allowed in silico virtual screening of inhibitors from databases, three of them being suitable in terms of absorption, distribution, metabolism, excretion, and toxicity (ADME/Tox) properties, which inhibited GMP (but not PMI) activity of the Xcc recombinant XanB protein in more than 50%. Inhibitors reduced citrus canker severity up to 95%, similarly to copper-based treatment. xanB is essential for Xcc pathogenicity, and XanB inhibitors can be used for the citrus canker control. IMPORTANCE: Xcc causes citrus canker, a threat to citrus production, which has been managed with copper, being required a more sustainable alternative for the disease control. XanB was previously found on the surface of Xcc, interacting with the host and displaying PMI and GMP activities. We demonstrated by xanB deletion and complementation that GMP activity plays a critical role in Xcc pathogenicity, particularly in biofilm formation. XanB homology modeling was performed, and in silico virtual screening led to carbohydrate-derived compounds able to inhibit XanB activity and reduce disease symptoms by 95%. XanB emerges as a promising target for drug design for control of citrus canker and other economically important diseases caused by Xanthomonas sp.

Discovery of Antibacterial Compounds against Xanthomonas citri subsp. citri from a Marine Fungus Aspergillus terreus SCSIO 41202 and the Mode of Action.

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a severe citrus disease. Currently, copper-containing pesticides are widely used to manage this disease, posing high risks to the environment and human health. This study reports the discovery of naturally occurring anti-Xcc compounds from a deep-sea fungus, Aspergillus terreus SCSIO 41202, and the possible mode of action. The ethyl acetate extract of A. terreus was subjected to bioassay-guided isolation, resulting in the discovery of eight anti-Xcc compounds (1-8) with minimum inhibitory concentrations (MICs) ranging from 0.078 to 0.625 mg/mL. The chemical structures of these eight metabolites were determined by integrative analysis of various spectroscopic data. Among these compounds, Asperporonin A (1) and Asperporonin B (2) were identified as novel compounds with a very unusual structural skeleton. The electronic circular dichroism was used to determine the absolute configurations of 1 and 2 through quantum chemical calculation. A bioconversion pathway involving pinacol rearrangement was proposed to produce the unusual compounds (1-2). Compound 6 exhibited an excellent anti-Xcc effect with a MIC value of 0.078 mg/mL, which was significantly more potent than the positive control CuSO4 (MIC = 0.3125 mg/mL). Compound 6 inhibited cell growth by disrupting biofilm formation, destroying the cell membrane, and inducing the accumulation of reactive oxygen species. In vivo tests indicated that compound 6 is highly effective in controlling citrus canker disease. These results indicate that compounds 1-8, especially 6, have the potential as lead compounds for the development of new, environmentally friendly, and efficient anti-Xcc pesticides.

The wall-associated receptor-like kinase CsWAKL01, positively regulated by the transcription factor CsWRKY53, confers resistance to citrus bacterial canker via regulation of phytohormone signaling.

Citrus bacterial canker (CBC) is a disease that poses a major threat to global citrus production and is caused by infection with Xanthomonas citri subsp. citri (Xcc). Wall-associated receptor-like kinase (WAKL) proteins play an important role in shaping plant resistance to various bacterial and fungal pathogens. In a prior report, CsWAKL01 was identified as a candidate Xcc-inducible gene found to be upregulated in CBC-resistant citrus plants. However, the functional role of CsWAKL01 and the mechanisms whereby it may influence resistance to CBC have yet to be clarified. Here, CsWAKL01 was found to localize to the plasma membrane, and the overexpression of the corresponding gene in transgenic sweet oranges resulted in the pronounced enhancement of CBC resistance, whereas its knockdown had the opposite effect. Mechanistically, the ability of CsWAKL01 was linked to its ability to reprogram jasmonic acid, salicylic acid, and abscisic acid signaling activity. CsWRKY53 was further identified as a transcription factor capable of directly binding the CsWAKL01 promoter and inducing its transcriptional upregulation. CsWRKY53 silencing conferred greater CBC susceptibility to infected plants. Overall, these data support a model wherein CsWRKY53 functions as a positive regulator of CsWAKL01 to enhance resistance to CBC via the reprogramming of phytohormone signaling. Together these results offer new insight into the mechanisms whereby WAKLs shape phytopathogen resistance while underscoring the potential value of targeting the CsWRKY53-CsWAKL01 axis when seeking to breed CBC-resistant citrus plant varieties.

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.

EnvC Homolog Encoded by Xanthomonas citri subsp. citri Is Necessary for Cell Division and Virulence.

Peptidoglycan hydrolases are enzymes responsible for breaking the peptidoglycan present in the bacterial cell wall, facilitating cell growth, cell division and peptidoglycan turnover. Xanthomonas citri subsp. citri (X. citri), the causal agent of citrus canker, encodes an Escherichia coli M23 peptidase EnvC homolog. EnvC is a LytM factor essential for cleaving the septal peptidoglycan, thereby facilitating the separation of daughter cells. In this study, the investigation focused on EnvC contribution to the virulence and cell separation of X. citri. It was observed that disruption of the X. citri envC gene (ΔenvC) led to a reduction in virulence. Upon inoculation into leaves of Rangpur lime (Citrus limonia Osbeck), the X. citri ΔenvC exhibited a delayed onset of citrus canker symptoms compared with the wild-type X. citri. Mutant complementation restored the wild-type phenotype. Sub-cellular localization confirmed that X. citri EnvC is a periplasmic protein. Moreover, the X. citri ΔenvC mutant exhibited elongated cells, indicating a defect in cell division. These findings support the role of EnvC in the regulation of cell wall organization, cell division, and they clarify the role of this peptidase in X. citri virulence.

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.

Design and Synthesis of Mandelic Acid Derivatives for Suppression of Virulence via T3SS against Citrus Canker.

Citrus canker, a highly contagious bacterial disease caused by Xanthomonas citri subsp. citri (Xcc), poses a substantial threat to citrus crops, leading to serious reductions in fruit yield and economic losses. Most commonly used bactericides against Xcc lead to the rapid development of resistant subpopulations. Therefore, it is imperative to create novel drugs, such as type III secretion system (T3SS) inhibitors, that specifically target bacterial virulence factors rather than bacterial viability. In our study, we designed and synthesized a series of mandelic acid derivatives including 2-mercapto-1,3,4-thiazole. Seven substances were found to reduce the level of transcription of hpa1 without affecting bacterial viability. In vivo bioassays indicated that compound F9 significantly inhibited hypersensitive response and pathogenicity. RT-qPCR assays showed that compound F9 visibly suppressed the expression of Xcc T3SS-related genes as well as citrus canker susceptibility gene CsLOB1. Furthermore, the combination with compound F9 and quorum-quenching bacteria HN-8 can also obviously alleviate canker symptoms.

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.