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Citrus canker, which is caused by Xanthomonas citri, is a severe disease that affects citrus plants worldwide. This paper aimed to compare, for the first time, the chemical composition and anti-Xanthomonas citri activities of essential oils from Schinus molle fresh and dry leaves (EO-FL and EO-DL, respectively). Anti-X. citri activity of spathulenol, the major constituent of oils, was also evaluated. Activities were screened by the broth microdilution method on 96‐well culture plates. Three major constituents were identified in EO-FL and EO-DL by GC-MS and GC-FID: spathulenol, β-caryophyllene and caryophyllene oxide. EO-DL (MIC = 31.25 µg/mL), EO-FL (MIC = 62.5 µg/mL) and spathulenol (MIC = 100 µg/mL) were active against X. citri strains (resistant, tolerant and sensitive to copper). Even though results showed that in vitro potential of EO-FL, EO-DL and spathulenol against X. citri, further in vivo studies are needed to prove their applicability to the biocontrol of citrus canker. [ABSTRACT FROM AUTHOR]

Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker, elicits canker symptoms in citrus plants because of the transcriptional activator-like (TAL) effector PthA4, which activates the expression of the citrus susceptibility gene CsLOB1. This study reports the regulation of the putative carbohydrate-binding protein gene Cs9g12620 by PthA4-mediated induction of CsLOB1 during Xcc infection. We found that the transcription of Cs9g12620 was induced by infection with Xcc in a PthA4-dependent manner. Even though it specifically bound to a putative TAL effector-binding element in the Cs9g12620 promoter, PthA4 exerted a suppressive effect on the promoter activity. In contrast, CsLOB1 bound to the Cs9g12620 promoter to activate its expression. The silencing of CsLOB1 significantly reduced the level of expression of Cs9g12620, which demonstrated that Cs9g12620 was directly regulated by CsLOB1. Intriguingly, PhtA4 interacted with CsLOB1 and exerted feedback control that suppressed the induction of expression of Cs9g12620 by CsLOB1. Transient overexpression and gene silencing revealed that Cs9g12620 was required for the optimal development of canker symptoms. These results support the hypothesis that the expression of Cs9g12620 is dynamically directed by PthA4 for canker formation through the PthA4-mediated induction of CsLOB1. [ABSTRACT FROM AUTHOR]

Introduction: Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a devastating disease worldwide. Previously, we successfully generated cankerresistant Citrus sinensis cv. Hamlin lines in the T0 generation. This was achieved through the transformation of embryogenic protoplasts using the ribonucleoprotein (RNP) containing Cas12a and one crRNA to edit the canker susceptibility gene, CsLOB1, which led to small indels. Methods: Here, we transformed embryogenic protoplasts of Hamlin with RNP containing Cas12a and three crRNAs. Results: Among the 10 transgene-free genome-edited lines, long deletions were obtained in five lines. Additionally, inversionswere observed in three of the five edited lines with long deletions, but not in any edited lines with short indel mutations, suggesting long deletions maybe required for inversions. Biallelic mutations were observed for each of the three target sites in four of the 10 edited lines when three crRNAs were used, demonstrating that transformation of embryogenic citrus protoplasts with Cas12a and three crRNAs RNP can be very efficient for multiplex editing. Our analysis revealed the absence of off-target mutations in the edited lines. These cslob1 mutant lines were canker-resistant and no canker symptoms were observed after inoculation with Xcc and Xcc growth was significantly reduced in the cslob1 mutant lines compared to the wild type plants. Discussion: Taken together, RNP (Cas12a and three crRNAs) transformation of embryogenic protoplasts of citrus provides a promising solution for transgene-free multiplex genome editing with high efficiency and for deletion of long fragments. [ABSTRACT FROM AUTHOR]

Citriculture has been one of the most important agricultural activities worldwide. Brazil is among the five major citrus producers in the world, together with China, the European Union (EU), the United States, and Mexico. Together, these five groups are responsible for about 50% of the global citrus fruit production and this industrial segment is responsible for generating direct and indirect jobs. However, several citric diseases such as citrus canker, huanglongbing, citrus variegated chlorosis, and citrus black spot have been associatedwith annual losses of tons of fruits and orange trees impacting the global economy for decades. Citrus canker is caused by theGram-negative bacteria Xanthomonas citri subsp. citri. and is associatedwith symptoms such as the formation of chlorotic rings in the leaves, stems, and fruits. Huanglongbing (HLB) is mainly associated with the Gram-negative bacteria Candidatus Liberibacter spp. and its main symptoms are the appearance of yellowish branches and deformed small leaves with yellowish spots. Citrus variegated chlorosis (CVC) is associated with the bacterium Xylella fast idiosa and causes chlorotic spots with irregular edges on leaves and deformation of newleaves. Citrus black spot (CBS) is caused by the fungus Phyllosticta citricarpa and generates lesions on fruits and reddish-brown leaf lesions. Since citrus is important for global agriculture, the current review addresses the main features of these important diseases includingtheir symptoms andtransmission, aswell as thediagnosis and control strategies that have been studied so far for application in the field. [ABSTRACT FROM AUTHOR]

Citrus canker disease, caused by Xanthomonas citri subsp. citri, poses a significant threat to global citrus production. The control of the disease in the field relies mainly on the use of conventional tools such as copper compounds, which are harmful to the environment and could lead to bacterial resistance. This scenario stresses the need for new and sustainable technologies to control phytopathogens, representing a key challenge in developing studies that translate basic into applied knowledge. During infection, X. citri subsp. Cltri secretes a transcriptional activator-like effector that enters the nucleus of plant cells, activating the expression of the canker susceptibility gene LATERAL ORGAN BOUNDARIES 1 (LOBA. In this study, we explored the use of antisense oligonucleotides (ASOs) with phosphorothioate modifications to transiently inhibit the gene expression of C.s LOBJ in Cirrus sinensis. We designed and validated three potential ASO sequences, which led to a significant reduction in disease symptoms compared with the control. The selected AS03-CsLOBI significantly decreased the expression level of CsLOBJ when delivered through two distinct delivery methods, and the reduction of the symptoms ranged from approximately 15 to 83%. Notably, plants treated with ASO3 did not exhibit an increase in symptom development over the evaluation period. This study highlights the efficacy of ASO technology, based on short oligonucleotide chemically modified sequences, as a promising tool for controlling phytopathogens without the need for genetic transformation or plant regeneration. Our results demonstrate the potential of ASOs as a biotechnological tool for the management of citrus canker disease. [ABSTRACT FROM AUTHOR]

Xanthomonas citri sub sp. citri (Xcc) causes citrus bacterial canker (CBC), a highly destructive disease of citrus plants globally. A comprehensive survey commenced in seven locations of District Sargodha, Pakistan, to determine the incidence of CBC on Kinnow, Feutrell’s early (Citrus reticulata Blanco), and Musambi (Citrus sinensis) cultivars. Additionally, 47 citrus cultivars reached assessment for their response to Xcc for disease severity. The highest incidence of CBC emerged in Kinnow at Shahpur (57.3%), Bhera (40.5%), and Sargodha (35.0%). Feutrell’s early showed a higher incidence rate at Shahpur (35.0%), Bhalwal (35.0%), and Sargodha (30.0%). Meanwhile, Musambi plants exhibited an incidence of 15.0% at Shahpur and Bhera and 13.3% at Bhalwal. Among the 47 cultivars, Jaffa, Valencia Late, Sanguinello, and Musambi (Citrus sinensis) demonstrated resistance to CBC, as their severity levels were <4.0%. Tracco N (20.3%) and rough lemon (22.0%) proved susceptible cultivars. However, a high susceptibility to canker disease was notable in Shamber grapefruit (31.0%) and Chakotra (Pommelo) (34.6%). Understanding the susceptibility of diverse citrus cultivars to CBC provides valuable insights for breeding programs and integrated management of the CBC disease. [ABSTRACT FROM AUTHOR]

The current investigation assessed the effectiveness of 30 citrus germplasms in combating citrus canker pathogenesis in Assam, North‐East India. Significant differences were observed among the examined citrus germplasms in terms of per cent disease severity and disease reduction against Xanthomonas citri pv. citri. Among the 30 different germplasms, eight germplasms, namely, Jora tenga (14.67%), Bonjora (15.58%), Holong tenga (15.75%), Latipes (17.34%), Patijora (17.75%), Birajora (18.50%), Cleopatra mandarin (20.00%) and Ada jamir (20.18%), exhibited highest disease resistance potential. The study recorded that the germplasm Latipes, which are resistant, had the highest total phenol content (21.26 mg/g dry weight), while the susceptible one, Grapefruit (7.85 mg/g dry weight), had the lowest total phenol content. Jora tenga exhibited the highest flavonoid content, measuring at 6.48 mg/g dry weight, whereas White pummelo had the lowest flavonoid content, measuring at 1.88 mg/g dry weight. Similarly, the highest alkaloid percentage was recorded in resistant germplasm, Adajamir (10.52%), while the lowest was in susceptible germplasm, Volkameriana (2.30%). The levels of total phenol, alkaloid and flavonoid contents exhibited a significant negative correlation with the per cent disease severity of citrus canker disease in both Pearson correlation and Spearmen correlation analysis. The current research on bioprospecting of citrus germplasms in relation to citrus canker pathogenesis has the potential to offer valuable insights into mitigating the transmission of bacterial pathogens and eliminating sources of infection by employing resistant cultivars and implementing approved doses of plant protection formulations. [ABSTRACT FROM AUTHOR]

Abstract Fruits play an important role in human life on our planet, since they supply a variety of essential services. One of the paramount crops in Pakistan is Citrus reticulata (Kinnow), which plays a vital role in the country's economy. The citrus crops are confronted with various challenges such as fungi, bacteria, nematodes, and viruses, all of which have adverse effects on the quality and yield of the fruits. Citrus canker, in particular, stands as the most fatal disease, affecting numerous citrus species worldwide, inflicting devastating consequences. The objective of this study was to evaluate the impact of citrus canker on the morphology, physiology of leaves, and the quality of citrus fruits. The research was conducted in four major citrus-producing tehsils of the Sargodha district. The study found significant differences in morphological and physiological traits between healthy fruits and those infected with citrus canker. Healthy fruits exhibited higher values in fruit weight (FW) of 149.02 g, peel thickness (PT) of 3.76 g, rag weight (RW) of 35.95 g, leaf area (LA) of 22.49 cm2, and juice weight compared to the citrus canker-infected fruits. The significant variations in fruit weight, juice weight, chlorophyll content, vitamin C content were present between healthy and diseased fruits and leaves. A biochemical study revealed that healthy fruits had greater levels of total soluble solids (TSS), TSS-acid ratio, vitamin C, and reducing power (RP), but citrus canker-infected fruits had a higher acidity percentage. The significant decreases in important morphological and physicochemical characteristics, emphasizing the necessity for immediate disease control techniques to safeguard the citrus sector and maintain food security.

Abstract Background Citrus canker caused by the bacterium Xanthomonas citri (Hasse 1915) is known as a dangerous disease that has serious economic impacts on citrus production in many tropical and subtropical countries. In Iran, Citrus canker disease has caused significant damage to various citrus fruits, particularly limes. Nowadays, employing antagonistic bacteria as an effective and environmentally friendly method for plant disease management has gained special importance. Results Citrus leaf samples were collected from different regions of Iran, and after isolating epiphytic bacteria, ex planta and in planta investigations concerning antagonistic activities of the isolates against Xanthomonas citri pathotypes A and A* were performed. A total of 94 epiphytic bacterial isolates were isolated from citrus aerial parts. Based on biochemical, physiological, morphological, and genotypic tests, it was determined that these bacteria belong to the Bacillus and Staphylococcus genera. The highest inhibition activity against the pathogenic bacterium was related to isolates D4 and D5. Using the molecular method and the resulting dendrogram, it was found that these isolates were most similar to Bacillus amyloliquefaciens. The present findings demonstrated that pathogenicity test on key lime leaves infected with X. citri pathotypes A and A*, along with the application of the biocontrol strain B. amyloliquefaciens, resulted in a significant reduction in the number of canker lesions. Conclusion The results strongly suggested that the identified antagonistic bacterial isolates hold promising potential as biocontrol agents for managing citrus bacterial canker disease.

Abstract 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.

Fruits play an important role in human life on our planet, since they supply a variety of essential services. One of the paramount crops in Pakistan is Citrus reticulata (Kinnow), which plays a vital role in the country's economy. The citrus crops are confronted with various challenges such as fungi, bacteria, nematodes, and viruses, all of which have adverse effects on the quality and yield of the fruits. Citrus canker, in particular, stands as the most fatal disease, affecting numerous citrus species worldwide, inflicting devastating consequences. The objective of this study was to evaluate the impact of citrus canker on the morphology, physiology of leaves, and the quality of citrus fruits. The research was conducted in four major citrus-producing tehsils of the Sargodha district. The study found significant differences in morphological and physiological traits between healthy fruits and those infected with citrus canker. Healthy fruits exhibited higher values in fruit weight (FW) of 149.02 g, peel thickness (PT) of 3.76 g, rag weight (RW) of 35.95 g, leaf area (LA) of 22.49 cm2, and juice weight compared to the citrus canker-infected fruits. The significant variations in fruit weight, juice weight, chlorophyll content, vitamin C content were present between healthy and diseased fruits and leaves. A biochemical study revealed that healthy fruits had greater levels of total soluble solids (TSS), TSS-acid ratio, vitamin C, and reducing power (RP), but citrus canker-infected fruits had a higher acidity percentage. The significant decreases in important morphological and physicochemical characteristics, emphasizing the necessity for immediate disease control techniques to safeguard the citrus sector and maintain food security. [ABSTRACT FROM AUTHOR]

In this review, we highlight studies in which whole-genome sequencing, comparative genomics, and population genomics have provided unprecedented insights into past and ongoing pathogen evolution. These include new understandings of the adaptive evolution of secretion systems and their effectors. We focus on Xanthonionas pathosystems that have seen intensive study and improved our understanding of pathogen emergence and evolution, particularly in the context of host specialization: citrus canker, bacterial blight of rice, and bacterial spot of tomato and pepper. Across pathosystems, pathogens appear to follow a pattern of bursts of evolution and diversification that impact host adaptation. There remains a need for studies on the mechanisms of host range evolution and genetic exchange among closely related but differentially host-specialized species and to start moving beyond the study of specific strain and host cultivar pairwise interactions to thinking about these pathosystems in a community context. [ABSTRACT FROM AUTHOR]

Citrus bacterial canker disease, caused by Xanthomonas citri subsp. citri (Xcc), poses a significant global threat to the citrus industry. Lateral organ boundaries 1 (Lob1) is confirmed as a citrus susceptibility gene that induces pathogenesis by interaction with the PthA4 effector of Xcc. Citron C-05 (Citrus medica) is a Citrus genotype resistant to Xcc. However, there is little information available on the regulation of Lob1 in resistant genotypes, which is important for the breeding of citrus cultivars resistant to canker disease. This study aimed to identify upstream regulatory factors of Lob1 in Citron C-05 and to investigate its function in disease resistance. 'Bingtang' sweet orange (C. sinensis), a susceptible genotype, was utilized as the control. cDNA yeast libraries of Xcc-induced Citron C-05 and 'Bingtang' sweet orange were constructed. The capacities of 'Bingtang' and Citron C-05 were 1.896 × 107 and 2.154 × 107 CFU, respectively. The inserted fragments ranged from 500 to 2000 bp with a 100% recombination rate. The promoter of Lob1 was segmented into two pieces and the P1 fragment from both genotypes was used to construct a bait yeast (PAbAi-CsLob1-P1; PAbAi-CmLob1-P1). Through library screening with the bait yeast, upstream regulators interacting with the Lob1-P1 promoter were identified and then validated using Y1H and dual-luciferase tests. The expression analysis of the three transcript factors indicated that RMA3 was upregulated by inoculation with Xcc in the resistant Citron C-05, but not in the susceptible sweet orange. The overexpression of CsRMA3 in 'Bingtang' sweet orange led to reduced canker symptoms, with a significantly lower pathogen density in the leaves following Xcc inoculation. When CmRMA3 was silenced by virus-induced gene silencing (VIGS) in Citron C-05, typical canker symptoms appeared on the CmRMA3-silenced leaves at 15 days post-inoculation with Xcc. Further expression analyses revealed that the CmRMA3 transcription factor suppressed the expression of Lob1. These results suggest that RMA3 participates in the resistant reaction of Citron C-05 to Xcc infection, and such a response might be in relation to its suppression of the expression of the pathogenic gene Lob1. [ABSTRACT FROM AUTHOR]

Background: Citrus canker caused by the bacterium Xanthomonas citri (Hasse 1915) is known as a dangerous disease that has serious economic impacts on citrus production in many tropical and subtropical countries. In Iran, Citrus canker disease has caused significant damage to various citrus fruits, particularly limes. Nowadays, employing antagonistic bacteria as an effective and environmentally friendly method for plant disease management has gained special importance. Results: Citrus leaf samples were collected from different regions of Iran, and after isolating epiphytic bacteria, ex planta and in planta investigations concerning antagonistic activities of the isolates against Xanthomonas citri pathotypes A and A* were performed. A total of 94 epiphytic bacterial isolates were isolated from citrus aerial parts. Based on biochemical, physiological, morphological, and genotypic tests, it was determined that these bacteria belong to the Bacillus and Staphylococcus genera. The highest inhibition activity against the pathogenic bacterium was related to isolates D4 and D5. Using the molecular method and the resulting dendrogram, it was found that these isolates were most similar to Bacillus amyloliquefaciens. The present findings demonstrated that pathogenicity test on key lime leaves infected with X. citri pathotypes A and A*, along with the application of the biocontrol strain B. amyloliquefaciens, resulted in a significant reduction in the number of canker lesions. Conclusion: The results strongly suggested that the identified antagonistic bacterial isolates hold promising potential as biocontrol agents for managing citrus bacterial canker disease. [ABSTRACT FROM AUTHOR]

Plant pathogens can alter the behavior of their insect vectors as well as their survival and reproduction. The African psyllid, Trioza erytreae, is one of the vectors of Huanglongbing, a citrus disease caused mainly by "Candidatus Liberibacter asiaticus" (CLas). The purpose of this study was to characterize the effects of CLas on the psyllid, T. erytreae using Citrus volkamerina plants as the study system. The study focused more specifically on the CLas effects prior to and after its acquisition by the psyllid T. erytreae. Our results did not support the hypothesis that CLas effects psyllid probing behavior prior to acquisition; few differences were observed between uninfected T. erytrea feeding on CLas‐infected versus control plants. On the other hand, compared to psyllids that had completed their development on control plants, the ones that had completed their development on a CLas‐infected plant exhibited changes in their behavior (greater velocity), physiology (smaller mass) and biochemistry (lower water and lipid content). Altogether, our results confirm the existence of a marked postacquisition effect on the vector locomotor behavior and a minor preacquisition effect of CLas on the vector behavior, which can be partially explained by physiological and biochemical changes. [ABSTRACT FROM AUTHOR]

Citrus canker, caused by the bacterium Xanthomonas citri subsp. citri, is one of the main threats to citrus fruit production. Several phenolic compounds active against X. citri have been described in recent years. Benzene‐1,2,4‐triol is a bio‐based phenolic compound that has shown high potential as a scaffold for the synthesis of new anti‐X. citri compounds. However, benzene‐1,2,4‐triol is prone to oxidative dimerization. We evaluated the antibacterial activity of benzene‐1,2,4‐triol, its oxidized dimers, and analogous compounds. Benzene‐1,2,4‐triol has a low inhibitory concentration against X. citri (0.05 mM) and is also active against other bacterial species. Spontaneous formation of benzenetriol dimers (e. g. by contact with oxygen in aqueous solution) reduced the antimicrobial activity of benzenetriol solutions. Dimers themselves displayed lower antibacterial activity and where shown to be more stable in solution. Unlike many other phenolic compounds with anti‐X. citri activity, benzene‐1,2,4‐triol does not act by membrane permeabilization, but seems to limit the availability of iron to cells. Benzene‐1,2,4‐triol is widely recognized as toxic – our results indicate that the toxicity of benzene‐1,2,4‐triol is largely due to spontaneously formed dimers. Stabilization of benzene‐1,2,4‐triol will be required to allow the safe use of this compound. [ABSTRACT FROM AUTHOR]

Pestalotioid fungi were isolated in pure culture from symptomatic plants of Callistemon laevis, C. viminalis, Luma apiculata (marketed as "Myrtus luma"), Myrtus communis subsp. tarentina, and M. communis var. 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. Neopestalotiopsis hispanica and N. rosae caused symptoms in all hosts of the present study, while the other Neopestalotiopsis species tested showed no symptoms on Luma apiculata. [ABSTRACT FROM AUTHOR]

BACKGROUND: Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is a devastating bacterial disease that reduces citrus yield and quality, posing a serious threat to the citrus industry. Several conventional chemicals have been used to control citrus canker. However, this approach often leads to the excessive use of chemical agents, can exacerbate environmental pollution and promotes the development of resistant Xcc. Therefore, there is significant interest in the development of efficient and environmentally friendly technologies to control citrus canker. RESULTS: In this study, water‐soluble ZnO quantum dots (ZnO QDs) were synthesised as an efficient nanopesticide against Xcc. The results showed that the antibacterial activity of ZnO QDs irradiated with visible light [half‐maximal effective concentration (EC50) = 33.18 μg mL−1] was ~3.5 times higher than that of the dark‐treated group (EC50 = 114.80 μg mL−1). ZnO QDs induced the generation of reactive oxygen species (•OH, •O−2 and 1O2) under light irradiation, resulting in DNA damage, cytoplasmic destruction, and decreased catalase and superoxide dismutase activities. Transcription analysis showed downregulation of Xcc genes related to 'biofilms, virulence, adhesion' and 'DNA transfer' exposure to ZnO QDs. More importantly, ZnO QDs also promoted the growth of citrus. CONCLUSION: This research provides new insights into the photocatalytic antibacterial mechanisms of ZnO QDs and supports the development of more efficient and safer ZnO QDs‐based nanopesticides to control citrus canker. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Citrus canker is a disease caused by the gram-negative bacterium Xanthomonas citri subp. citri (X. citri), which affects all commercially important varieties of citrus and can lead to significant losses. Fruit sanitization with products such as chlorine-based ones can reduce the spread of the disease. While effective, their use raises concerns about safety of the workers. This work proposes essential oils (EOs) as viable alternatives for fruit sanitization. EOs from Cymbopogon species were evaluated as to their antibacterial activity, their effect on the bacterial membrane, and their ability to sanitize citrus fruit. The in vitro assays revealed that the EOs from C. schoenanthus and C. citratus had a lower bactericidal concentration at 312 mg L−1, followed by 625 mg L−1 for C. martini and C. winterianus. Microscopy assay revealed that the bacterial cell membranes were disrupted after 15 min of contact with all EOs tested. Regarding the sanitizing potential, the EOs with higher proportions of geraniol were more effective in sanitizing acid limes. Fruit treated with C. shoenanthus and C. martini showed a reduction of ∼68% in the recovery of viable bacterial cells. Therefore, these EOs can be used as viable natural alternatives in citrus fruit disinfection. [ABSTRACT FROM AUTHOR]

Wolbachia, a group of Gram-negative symbiotic bacteria, infects nematodes and a wide range of arthropods. Diaphorina citri Kuwayama, the vector of Candidatus Liberibacter asiaticus (CLas) that causes citrus greening disease, is naturally infected with Wolbachia (wDi). However, the interaction between wDi and D. citri remains poorly understood. In this study, we performed a pan-genome analysis using 65 wDi genomes to gain a comprehensive understanding of wDi. Based on average nucleotide identity (ANI) analysis, we classified the wDi strains into Asia and North America strains. The ANI analysis, principal coordinates analysis (PCoA), and phylogenetic tree analysis supported that the D. citri in Florida did not originate from China. Furthermore, we found that a significant number of core genes were associated with metabolic pathways. Pathways such as thiamine metabolism, type I secretion system, biotin transport, and phospholipid transport were highly conserved across all analyzed wDi genomes. The variation analysis between Asia and North America wDi showed that there were 39,625 single-nucleotide polymorphisms (SNPs), 2153 indels, 10 inversions, 29 translocations, 65 duplications, 10 SV-based insertions, and 4 SV-based deletions. The SV-based insertions and deletions involved genes encoding transposase, phage tail tube protein, ankyrin repeat (ANK) protein, and group II intron-encoded protein. Pan-genome analysis of wDi contributes to our understanding of the geographical population of wDi, the origin of hosts of D. citri, and the interaction between wDi and its host, thus facilitating the development of strategies to control the insects and huanglongbing (HLB). [ABSTRACT FROM AUTHOR]

Background: Pakistan’s economy largely depends on citrus cultivation, and citrus fruits generate significant foreign exchange. Xanthomonas citri pv. citri (Xcc) is the primary cause of citrus canker (CC), which poses a significant threat to the industry. The management of disease is made more difficult by the lack of resistant variants against different Xcc races. Understanding and addressing Xcc are critical for maintaining the industry given the economic reliance on citrus. Methods: In order to isolate Xcc in a lab, a thorough survey was conducted in the districts of Bahawalpur, Multan, and Dera Ghazi Khan to collect samples showing canker symptoms. Gram-negative bacteria were identified in the isolates by biochemical analysis, and Koch’s postulates confirmed Xcc as the CC causing agent. DNA extraction and sequencing were used in the molecular characterization, which confirmed the phylogenetic relationship with Xcc. Using the disc sensitivity method, nine antibiotics were tested at 300, 500, and 700 ppm concentrations to evaluate CC management. Amoxicillin was found to be highly effective in inhibiting the growth of Xcc colonies. Results: The study provided morphogenetic insights and established Xcc as the causal agent of CC. All strains matched Xcc in molecular characterization, but antibiotic sensitivity testing revealed inconsistent efficacy. Amoxicillin proved to be very effective at stopping the growth of Xcc colonies at every tested dosage. Conclusion: This study makes an important contribution to our understanding of XCC and helps the citrus industry develop better disease control plans. Future interventions against citrus canker can benefit greatly from the understanding provided by morphogenetic characterization and antibiotic sensitivity profiles. The discovery of amoxicillin’s high efficacy highlights the drug’s potential for treating diseases linked to Xcc. These results help to protect citrus growing, maintaining the viability of an important industry in Pakistan’s agricultural landscape.

Abstract 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.

Abstract Virus-induced gene silencing (VIGS) is a powerful technology for molecular characterization of gene functions in plants and has the potential to prevent and control plant diseases. Unfortunately, VIGS in many woody perennials such as citrus are severely hindered because they have a long juvenile period and are recalcitrant to infection compared to herbaceous plants. Here, we report the development of a simple and effective VIGS system based on citrus leaf blotch virus (CLBV), a virus endemic in most citrus-producing countries but showing no symptoms in most citrus varieties. By Agrobacterium-mediated vacuum-infiltration (AVI), pCLBV201-su245 and pCLBV201-pds391 triggered efficient gene silencing (85%) and silencing phenotypes began to appear at 30 days post infiltration/inoculation (dpi), a similar period for achieving that on herbaceous plants. Moreover, the silencing phenotypes could still be observed four years post inoculation. Further, pCLBV201-lob369 was constructed to silence the gene lateral organ boundaries1 (lob1), encoding a key factor for susceptibility to citrus canker that caused by Xanthomonas citri ssp. citri (Xcc). The resistance evaluation results showed that the lesion area, disease index, and bacterial content in the pCLBV201-lob369 treatment group were decreased by 64%, 14%, and 67%, respectively. This work provides a simple and effective VIGS system for citrus which has the potential to be used for diseases control.

SUMMARY: Citrus bacterial canker (CBC) is a serious bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) that adversely impacts the global citrus industry. In a previous study, we demonstrated that overexpression of an Xcc‐inducible apetala 2/ethylene response factor encoded by Citrus sinensis, CsAP2‐09, enhances CBC resistance. The mechanism responsible for this effect, however, is not known. In the present study, we showed that CsAP2‐09 targeted the promoter of the Xcc‐inducible WRKY transcription factor coding gene CsWRKY25 directly, activating its transcription. CsWRKY25 was found to localize to the nucleus and to activate transcriptional activity. Plants overexpressing CsWRKY25 were more resistant to CBC and showed higher expression of the respiratory burst oxidase homolog (RBOH) CsRBOH2, in addition to exhibiting increased RBOH activity. Transient overexpression assays in citrus confirmed that CsWRKY25 and CsRBOH2 participated in the generation of reactive oxygen species (ROS) bursts, which were able to restore the ROS degradation caused by CsAP2‐09 knockdown. Moreover, CsWRKY25 was found to bind directly to W‐box elements within the CsRBOH2 promoter. Notably, CsRBOH2 knockdown had been reported previously to reduce the CBC resistance, while demonstrated in this study, CsRBOH2 transient overexpression can enhance the CBC resistance. Overall, our results outline a pathway through which CsAP2‐09‐CsWRKY25 transcriptionally reprograms CsRBOH2‐mediated ROS homeostasis in a manner conducive to CBC resistance. These data offer new insight into the mechanisms and regulatory pathways through which CsAP2‐09 regulates CBC resistance, highlighting its potential utility as a target for the breeding of CBC‐resistant citrus varieties. Significant Statement: In the present study, we identify a pathway in which CsAP2‐09 and CsWRKY25 form a transcription factor cascade, transcriptionally reprogramming CsRBOH2‐mediated reactive oxygen species homeostasis and citrus bacterial canker (CBC) resistance. Our study explores new insights into the mechanistic role of CsAP2‐09 and the associated regulatory network, extending the potential value of CsAP2‐09 in the breeding of CBC‐tolerant citrus plants. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Citrus canker disease affects citrus production. This disease is caused by Xanthomonas citri subsp. citri (Xcc). Previous studies confirmed that during Xcc infection, PthA4, a transcriptional activator like effector (TALE), is translocated from the pathogen to host plant cells. PthA4 binds to the effector binding elements (EBEs) in the promoter region of canker susceptibility gene LOB1 (EBEPthA4-LOBP) to activate its expression and subsequently cause canker symptoms. Previously, the Cas12a/CBE co-editing method was employed to disrupt EBEPthA4-LOBP of pummelo, which is highly homozygous. However, most commercial citrus cultivars are heterozygous hybrids and more difficult to generate homozygous/biallelic mutants. Here, we employed Cas12a/CBE co-editing method to edit EBEPthA4-LOBP of Hamlin (Citrus sinensis), a commercial heterozygous hybrid citrus cultivar grown worldwide. Binary vector GFPp1380N-ttLbCas12a:LOBP1-mPBE:ALS2:ALS1 was constructed and shown to be functional via Xcc-facilitated agroinfiltration in Hamlin leaves. This construct allows the selection of transgene-free regenerants via GFP, edits ALS to generate chlorsulfuron-resistant regenerants as a selection marker for genome editing resulting from transient expression of the T-DNA via nCas9-mPBE:ALS2:ALS1, and edits gene(s) of interest (i.e., EBEPthA4-LOBP in this study) through ttLbCas12a, thus creating transgene-free citrus. Totally, 77 plantlets were produced. Among them, 8 plantlets were transgenic plants (#HamGFP1 - #HamGFP8), 4 plantlets were transgene-free (#HamNoGFP1 - #HamNoGFP4), and the rest were wild type. Among 4 transgene-free plantlets, three lines (#HamNoGFP1, #HamNoGFP2 and #HamNoGFP3) contained biallelic mutations in EBEpthA4, and one line (#HamNoGFP 4) had homozygous mutations in EBEpthA4. We achieved 5.2% transgene-free homozygous/biallelic mutation efficiency for EBEPthA4-LOBP in C. sinensis cv. Hamlin, compared to 1.9% mutation efficiency for pummelo in a previous study. Importantly, the four transgenefree plantlets and 3 transgenic plantlets that survived were resistant against citrus canker. Taken together, Cas12a/CBE co-editing method has been successfully used to generate transgene-free canker-resistant C. sinensis cv. Hamlin in the T0 generation via biallelic/homozygous editing of EBEpthA4 of the canker susceptibility gene LOB1. [ABSTRACT FROM AUTHOR]

Aims Citrus canker caused by Xanthomonas citri subsp. citri (X. citri) is a disease of economic importance. Control of this disease includes the use of metallic copper, which is harmful to the environment and human health. Previous studies showed that the crude extract from the fungus Pseudogymnoascus sp. LAMAI 2784 isolated from Antarctic soil had in vitro antibacterial action against X. citri. The aim of the present study was to expand the applications of this extract. Methods and results In greenhouse assays, the crude extract was able to reduce bacterial infection on citrus leaves from 1.55 lesions/cm2 (untreated plants) to 0.04 lesions/cm2. Bisdechlorogeodin was identified as the main compound of the bioactive fraction produced by Pseudogymnoascus sp. LAMAI 2784, which inhibited bacterial growth in vitro (IC90 ≈ 156 µg ml−1) and permeated 80% of X. citri cells, indicating that the membrane is the primary target. Conclusion The present results showed that the bioactive fraction of the extract is mainly composed of the compound bisdechlorogeodin, which is likely responsible for the biological activity against X. citri , and the main mechanism of action is the targeting of the cell membrane. This study indicates that bisdechlorogeodin has valuable potential for the control of X. citri. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Virus-induced gene silencing (VIGS) is a powerful technology for molecular characterization of gene functions in plants and has the potential to prevent and control plant diseases. Unfortunately, VIGS in many woody perennials such as citrus are severely hindered because they have a long juvenile period and are recalcitrant to infection compared to herbaceous plants. Here, we report the development of a simple and effective VIGS system based on citrus leaf blotch virus (CLBV), a virus endemic in most citrus-producing countries but showing no symptoms in most citrus varieties. By Agrobacterium-mediated vacuum-infiltration (AVI), pCLBV201-su245 and pCLBV201-pds391 triggered efficient gene silencing (85%) and silencing phenotypes began to appear at 30 days post infiltration/inoculation (dpi), a similar period for achieving that on herbaceous plants. Moreover, the silencing phenotypes could still be observed four years post inoculation. Further, pCLBV201-lob369 was constructed to silence the gene lateral organ boundaries1 (lob1), encoding a key factor for susceptibility to citrus canker that caused by Xanthomonas citri ssp. citri (Xcc). The resistance evaluation results showed that the lesion area, disease index, and bacterial content in the pCLBV201-lob369 treatment group were decreased by 64%, 14%, and 67%, respectively. This work provides a simple and effective VIGS system for citrus which has the potential to be used for diseases control. [ABSTRACT FROM AUTHOR]

Citrus bacterial canker (CBC) is a severe bacterial infection caused by Xanthomonas citri subsp. citri (Xcc), which continues to adversely impact citrus production worldwide. Members of the GATA family are important regulators of plant development and regulate plant responses to particular stressors. This report aimed to systematically elucidate the Citrus sinensis genome to identify and annotate genes that encode GATAs and evaluate the functional importance of these CsGATAs as regulators of CBC resistance. In total, 24 CsGATAs were identified and classified into four subfamilies. Furthermore, the phylogenetic relationships, chromosomal locations, collinear relationships, gene structures, and conserved domains for each of these GATA family members were also evaluated. It was observed that Xcc infection induced some CsGATAs, among which CsGATA12 was chosen for further functional validation. CsGATA12 was found to be localized in the nucleus and was differentially upregulated in the CBC-resistant and CBC-sensitive Kumquat and Wanjincheng citrus varieties. When transiently overexpressed, CsGATA12 significantly reduced CBC resistance with a corresponding increase in abscisic acid, jasmonic acid, and antioxidant enzyme levels. These alterations were consistent with lower levels of salicylic acid, ethylene, and reactive oxygen species. Moreover, the bacteria-induced CsGATA12 gene silencing yielded the opposite phenotypic outcomes. This investigation highlights the important role of CsGATA12 in regulating CBC resistance, underscoring its potential utility as a target for breeding citrus varieties with superior phytopathogen resistance. [ABSTRACT FROM AUTHOR]

The development of efficient and environmentally friendly plant protection systems is one of the major challenges for a sustainable agriculture of the future. Citrus canker caused by the pathogen Xanthomonas citri subsp. citri (X. citri) affects all cultivated citrus species worldwide and is responsible for enormous economic losses and restrictions in international trade. Currently used commercial copper‐based formulations are the origin of contamination for soil and groundwater, affecting local ecosystems and human health. A copper‐free sustainable microgel‐based plant protection system able to efficiently combat X. citri is developed. Microgels decorated with anchor peptides exhibit strong non‐covalent attachment to the surface of orange leaves and have the ability to release hexyl gallate, which inhibits the growth and spreading of pathogens. The tailored design of the microgel network allows high loadings of hexyl gallate (up to 40 wt.%) and a controlled release of gallates from microgels that provide long‐term protection. The antibacterial activity of the hexyl gallate‐loaded microgels is demonstrated by various in vitro assays as well as on orange plants in greenhouse settings. The experimental results suggest that the developed sustainable microgel‐based plant protection system for citrus trees allows efficient abatement of X. citri and reduces environmental pollution caused by copper formulations. [ABSTRACT FROM AUTHOR]

Introduction: Asian citrus psyllid (ACP, Diaphorina citri) is an important transmission vector of "Candidatus Liberibacter asiaticus" (CLas), the causal agent of Huanglongbing (HLB), the most destructive citrus disease in the world. As there are currently no HLB-resistant rootstocks or varieties, the control of ACP is an important way to prevent HLB. Some viruses of insect vectors can be used as genetically engineered materials to control insect vectors. Methods: To gain knowledge on viruses in ACP in China, the prevalence of five RNA and DNA viruses was successfully determined by optimizing reverse transcription polymerase chain reaction (RT-PCR) in individual adult ACPs. The five ACPassociated viruses were identified as follows: diaphorina citri bunyavirus 2, which was newly identified by high-throughput sequencing in our lab, diaphorina citri reovirus (DcRV), diaphorina citri picorna-like virus (DcPLV), diaphorina citri bunyavirus (DcBV), and diaphorina citri densovirus-like virus (DcDV). Results: DcPLV was the most prevalent and widespread ACP-associated virus, followed by DcBV, and it was detected in more than 50% of all samples tested. DcPLV was also demonstrated to propagate vertically and found more in salivary glands among different tissues. Approximately 60% of all adult insect samples were co-infected with more than one insect pathogen, including the five ACPassociated viruses and CLas. Discussion: This is the first time these viruses, including the newly identified ACPassociated virus, have been detected in individual adult ACPs from natural populations in China's five major citrus-producing provinces. These results provide valuable information about the prevalence of ACP-associated viruses in China, some of which have the potential to be used as biocontrol agents. In addition, analysis of the change in prevalence of pathogens in a single insect vector is the basis for understanding the interactions between CLas, ACP, and insect viruses. [ABSTRACT FROM AUTHOR]

Citrus canker is an infectious bacterial disease and one of the major threats to the orange juice industry, a multibillion-dollar market that generates hundreds of thousands of jobs worldwide. This disease is caused by the Gram-negative bacterium Xanthomonas citri subsp. citri. In Brazil, the largest producer and exporter of concentrate orange juice, the control of citrus canker is exerted by integrated management practices, in which cupric solutions are intensively used in the orchards to refrain bacterial spreading. Copper ions accumulate and are as heavy metals toxic to the environment. Therefore, the aim of the present work was to evaluate bifunctional fusion proteins (BiFuProts) as novel and bio-/peptide-based alternatives to copper formulations to control citrus canker. BiFuProts are composed of an anchor peptide able to bind to citrus leaves, and an antimicrobial "killer" peptide to protect against bacterial infections of plants. The selected BiFuProt (Mel-CgDEF) was bactericidal against X. citri at 125 μg mL−1, targeting the bacterial cytoplasmic membrane within the first minutes of contact. The results in the greenhouse assays proved that Mel-CgDEF at 250 μg mL−1 provided protection against X. citri infection on the leaves, significantly reducing the number of lesions by area when compared with the controls. Overall, the present work showed that the BiFuProt Mel-CgDEF is a biobased and biodegradable possible alternative for substitute cupric formulations. Key points: • The bifunctional fusion protein Mel-CgDEF was effective against Xanthomonas citri. • Mel-CgDEF action mechanism was the disruption of the cytoplasmic membrane. • Mel-CgDEF protected citrus leaves against citrus canker disease. [ABSTRACT FROM AUTHOR]

Poplar is a major afforestation species which is widely planted worldwide. With the continuous expansion of poplar plant areas, the occurrence of poplar canker disease increased rapidly, causing serious damage to the trees. Currently, the major controlling method for poplar canker is chemical agents, but side effects such as environmental pollution and pathogen resistance are becoming more and more serious. There is an urgent need for developing effective non-polluting means to control the canker disease. By using microbiomes as a research tool, beneficial microorganisms that enhance plant disease resistance can be obtained. These antagonistic bacteria can inhibit the growth of canker pathogens without environmental pollution, and have good biological-control potential. This review mainly explores the role and mechanisms of antagonistic bacteria in the control of poplar canker, including inhibiting the growth of pathogenic bacteria through nutrient and water competition, secreting bacteriostatic substances, improving plant disease resistance to resist pathogen infection, and improving the host's own reactive oxygen species tolerance, in order to provide a theoretical basis for the development of efficient and specific bacterial agents, and scientific prevention and control technology. [ABSTRACT FROM AUTHOR]

The losses associated with the bacterial diseases of citrus leaves and fruits have been reported throughout all of Pakistan's citrus-growing regions and a lot of work was done on the morphological characterization of pre and post-harvest pathogens. However, this issue has not been addressed in Khanpur, which is a major citrus-producing city in Pakistan and can help in the economic growth of the country. The citrus fruits in the area under study exhibit disease symptoms during pre and post-harvesting, but the actual disease agents have not yet been identified. The retrospective object of the current research is to study the pathogenic variation along with the investigation of bacterial disease incidence i.e. citrus canker, bacterial spots, & HLB in citrus fruit. During a survey conducted in November 2019 and 2020, bacterial isolates from symptomatic citrus fruits were obtained from randomly chosen orchards in Khanpur.The disease assessment was estimated by determining prevalence, severity, and disease incidence (DI). The bacterial isolates were identified morphologically and further through 16S rRNA gene amplification. The identified species were Pseudomonas syringae, Pseudomonas viridiflava, Serratia marcescens, Xanthomonas axonopodis pv citri, and Candidatus liberibacter. In the pathogenicity test, the Xanthomonas strains responsible for the canker disease caused the most severe disease symptoms. To our knowledge, this is the first report on pathogens of citrus fruits in Khanpur. Given the significant contribution of citrus fruits to household income and foreign exchange in Pakistan, this study illustrates the molecular-based association of bacterial pathogens of citrus and the toxic behavior of pathogens which helps to provide recommendations on genetic testing and to develop management programs for disease management. The molecular identification of citrus pathogens will empower the citrus growers, researchers, and regulatory agencies to make informed decisions that will protect citrus crops, reduce economic losses, and promote sustainable agriculture. It is also likely to play an increasingly vital role in the future of citrus cultivation..

ABSTRACT 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.IMPORTANCEXcc 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.

The current study examined the correlation between the prevalence of Xanthomonas axonopodis L. and flavonoid content in the leaves and pulp of various citrus cultivars (both healthy and Xac-infected) across different regions of Pakistan. The study considered environmental factors such as temperature (°C), relative humidity (%), total precipitation (mm), and wind speed (km/h). Flavonoid levels were quantified using liquid chromatography-mass spectrometry. Statistical analysis revealed the presence of 12 different flavonoids in the citrus cultivars. The concentrations of these flavonoids were found to be lower in Xac-infected plants compared to healthy ones. Additionally, the amounts of each flavonoid varied under different climatic conditions. The findings of this study could be utilized to promote the commercialization of citrus varieties that are either resistant to Xac infection or possess high flavonoid content. [ABSTRACT FROM AUTHOR]

Citrus fruits, that belong to the Rutaceae. family, have a great economic, nutritional, and therapeutic importance. It has long been a significant source of foreign exchange for Pakistan. Unfortunately, various diseases affect its production and quality, the deadliest of which is known as citrus canker caused by Xanthomonas axonopodis pv. citri. The goal of current research was to isolate and characterize the strains causing citrus canker on the basis of phenotypical and genotypical features. Fruits with possible canker lesions were used for the isolation from the different Citrus Orchards in Khanpur, District Haripur, Pakistan. Citrus fruits, both symptomatic and asymptomatic, were sampled and evaluated during the survey. Only diseased citrus fruits yielded Xanthomonas-like bacterial strains were isolated and identified by carrying out different biochemical tests including catalase, Kovac's oxidase and KOH test. The isolated strains were further identified through polymerase chain reaction (PCR) by using specific ITS primers. Total of eight bacterial isolates were identified to be Xanthomonas spp. through biochemical tests and molecular results revelled the 2A and 2B bacterial isolates as., Xanthomonas citri subsp. citri. (Xcc). Thus, it was concluded that isolated strains were pathogenic bacteria, that is an actual cause of bacterial canker in commercial citrus fruits. Our findings suggested that diseased citrus fruits, could act as a source of dispersal for citrus canker in areas like Pakistan. [ABSTRACT FROM AUTHOR]

Xanthomonas citri pv. citri (Xcc) causes the devastating citrus canker disease. Xcc is known to have been introduced into Florida, USA in at least three different events in 1915, 1986 and 1995 with the first two claimed to be eradicated. It was questioned whether the Xcc introduction in 1986 has been successfully eradicated. Furthermore, it is unknown how Xcc has spread throughout the citrus groves in Florida. In this study, we investigated the population structure of Xcc to address these questions. We sequenced the whole genome of 343 Xcc strains collected from Florida groves between 1997 and 2016. Our analysis revealed two distinct clusters of Xcc. Our data strongly indicate that the claimed eradication of the 1986 Xcc introduction was not successful and Xcc strains from 1986 introduction were present in samples from at least 8 counties collected after 1994. Importantly, our data revealed that the Cluster 2 strains, which are present in all 20 citrus-producing counties sampled in Florida, originated from the Xcc introduction event in the Miami area in 1995. Our data suggest that Polk County is the epicenter of the dispersal of Cluster 2 Xcc strains, which is consistent with the fact that three major hurricanes passed through Polk County in 2004. As copper-based products have been extensively used to control citrus canker, we also investigated whether Xcc strains have developed resistance to copper. Notably, none of the 343 strains contained known copper resistance genes. Twenty randomly selected Xcc strains displayed sensitivity to copper. Overall, this study provides valuable insights into the introduction, eradication, spread, and copper resistance of Xcc in Florida. Author summary: Sequencing and analyzing the genomes of 343 Xcc strains collected from 1997 to 2016 have enabled us to gain a deeper understanding of its introduction, spread, and eradication. This information is critical for optimizing strategies for responding to the invasion of quarantine pathogens. We have conclusively demonstrated that the Xcc introduction in 1986 was not successfully eradicated, three major hurricanes in 2004 play a critical role in spreading Xcc throughout Florida, and Polk County is the epicenter of Xcc dispersal. Additionally, none of the tested Xcc strains displayed resistance to copper, consistent with the absence of copper resistance genes in their genome. [ABSTRACT FROM AUTHOR]

Diseases pose a significant threat to the citrus industry, and the accurate detection of these diseases represent key factors for their early diagnosis and precise control. Existing diagnostic methods primarily rely on image models trained on vast datasets and limited their applicability due to singular backgrounds. To devise a more accurate, robust, and versatile model for citrus disease classification, this study focused on data diversity, knowledge assistance, and modal fusion. Leaves from healthy plants and plants infected with 10 prevalent diseases (citrus greening, citrus canker, anthracnose, scab, greasy spot, melanose, sooty mold, nitrogen deficiency, magnesium deficiency, and iron deficiency) were used as materials. Initially, three datasets with white, natural, and mixed backgrounds were constructed to analyze their effects on the training accuracy, test generalization ability, and classification balance. This diversification of data significantly improved the model’s adaptability to natural settings. Subsequently, by leveraging agricultural domain knowledge, a structured citrus disease features glossary was developed to enhance the efficiency of data preparation and the credibility of identification results. To address the underutilization of multimodal data in existing models, this study explored semantic embedding methods for disease images and structured descriptive texts. Convolutional networks with different depths (VGG16, ResNet50, MobileNetV2, and ShuffleNetV2) were used to extract the visual features of leaves. Concurrently, TextCNN and fastText were used to extract textual features and semantic relationships. By integrating the complementary nature of the image and text information, a joint learning model for citrus disease features was achieved. ShuffleNetV2 + TextCNN, the optimal multimodal model, achieved a classification accuracy of 98.33% on the mixed dataset, which represented improvements of 9.78% and 21.11% over the single-image and single-text models, respectively. This model also exhibited faster convergence, superior classification balance, and enhanced generalization capability, compared with the other methods. The image-text multimodal feature fusion network proposed in this study, which integrates text and image features with domain knowledge, can identify and classify citrus diseases in scenarios with limited samples and multiple background noise. The proposed model provides a more reliable decision-making basis for the precise application of biological and chemical control strategies for citrus production. [ABSTRACT FROM AUTHOR]

In Mexico, Huanglongbing infection caused 42 and 33% decreases in orange and lemon production, respectively. Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is a vector of Candidatus Liberibacter bacterium that causes the disease. Commercial orchards were searched to identify entomopathogenic fungi of D. citri in the citrus zone of Huasteca Potosina, San Luis Potosi, and Llera, Tamaulipas, Mexico. Entomopathogenic fungi associated with Asian citrus psyllid adults are in three genera: Hirsutella citriformis (88%), Beauveria bassiana (2%), and Sporothrix insectorum (10%). This is the first report of Sporothrix insectorum parasitizing H. citriformis in México. [ABSTRACT FROM AUTHOR]

Citrus canker caused by Xanthomonas citri subsp. citri is a devastating bacterial disease with severe implications for the citrus industry. Microorganisms possessing biocontrol capabilities against X. citri subsp. citri offer a highly promising strategy for healthy citrus management. In the present study, a broad-spectrum antagonist strain ZJLMBA1908 with potent antibacterial activity against X. citri subsp. citri was isolated from symptomatic lemon leaves, and identified as Bacillus amyloliquefaciens. Cell-free supernatant (CFS) of strain ZJLMBA1908 also exhibited remarkable antimicrobial activity, especially suppressing the growth of X. citri subsp. citri and Nigrospora oryzae, with inhibition rates of 27.71% and 63.75%, respectively. The antibacterial crude extract (CE) derived from the CFS displayed effective activity against X. citri subsp. citri. A preventive treatment using the CE significantly reduced the severity and incidence of citrus canker in a highly susceptible citrus host. Additionally, the CE maintained activity in the presence of protease and under a wide range of temperature and pH treatments. Applying high-performance liquid chromatography (HPLC) to separate and purify the CE resulted in the discovery of one highly potent anti-X. citri subsp. citri subfraction, namely CE3, which could completely inhibit the growth of X. citri subsp. citri. Liquid chromatography–electrospray ionization–mass spectrometry (LC–ESI–MS) analysis revealed that CE3 mainly consisted of palmitic acid, surfactin C15, phytosphingosine and dihydrosphingosine. Taken together, the results contribute to the possible biocontrol mechanisms of B. amyloliquefaciens ZJLMBA1908, as well as providing a promising new candidate strain as a biological control agent for controlling citrus canker. [ABSTRACT FROM AUTHOR]

BACKGROUND: Citrus canker and citrus blue mold are two severe diseases in citrus plants, which are mainly caused by Xanthomonas citri susp. citri (Xcc) and Penicillium italicum, respectively. The currently widely used pesticides for these two diseases are harmful to human health and the environment. Therefore, searching for novel antimicrobial agents, especially from natural resources, is getting increasing interest. RESULTS: In this study, the crude extract of Streptomyces sp. GLL‐9, an endophyte from a navel orange tree, was found to exhibit excellent antimicrobial effects against Xcc and P. italicum. Bioassay‐guided isolation led to the discovery of three actinomycins (Acts), actinomycin X2 (Act‐X2), actinomycin D (ActD), and actinomycin XOβ (Act‐XOβ). The MIC (minimum inhibitory concentration) values of Act‐X2, ActD, and Act‐XOβ were 31.25, 62.50, and 62.50 μg mL−1 against Xcc, respectively, while 62.50 (Act‐X2) and 125.00 μg mL−1 (ActD) against P. italicum, being better or comparable to the positive controls. The highest yield of Acts was obtained by solid‐state fermentation with rice containing 1% L‐tryptophan as a culture medium, being 6.03, 3.07, and 1.02 mg g−1, for Act‐X2, ActD, and Act‐XOβ, respectively. The ethyl acetate extract of Streptomyces sp. GLL‐9 cultivated under the optimal fermentation conditions (EAE‐1) can efficiently control these two citrus diseases by excessively producing reactive oxygen species (ROS) in both pathogens, damaging the cell membranes of P. italicum, and inhibiting the growth of Xcc. In addition, Act‐X2, ActD, and EAE‐1 displayed broad‐spectrum antifungal activity. CONCLUSION: EAE‐1 and Acts produced by Streptomyces sp. GLL‐9 have high potential as novel antimicrobial agents against plant pathogens. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

This document, titled "PP 1/122 (3) Phytophthora nicotianae var. parasitica and other Phytophthora species causing canker on citrus," provides guidelines for conducting trials to evaluate the efficacy of fungicides against Phytophthora species that cause canker on citrus trees. The document outlines the experimental conditions, trial conditions, design and layout of the trial, application of treatments, mode of assessment, recording and measurements, and reporting of results. It emphasizes the importance of following good experimental practices and provides references to other relevant standards. The document does not provide specific results or conclusions, but rather serves as a guide for researchers conducting trials on this topic. [Extracted from the article]

Summary: Citrus bacterial canker (CBC), caused by Xanthomonas citri subsp. citri (Xcc), causes dramatic losses to the citrus industry worldwide. Transcription activator‐like effectors (TALEs), which bind to effector binding elements (EBEs) in host promoters and activate transcription of downstream host genes, contribute significantly to Xcc virulence. The discovery of the biochemical context for the binding of TALEs to matching EBE motifs, an interaction commonly referred to as the TALE code, enabled the in silico prediction of EBEs for each TALE protein. Using the TALE code, we engineered a synthetic resistance (R) gene, called the Xcc‐TALE‐trap, in which 14 tandemly arranged EBEs, each capable of autonomously recognizing a particular Xcc TALE, drive the expression of Xanthomonas avrGf2, which encodes a bacterial effector that induces plant cell death. Analysis of a corresponding transgenic Duncan grapefruit showed that transcription of the cell death‐inducing executor gene, avrGf2, was strictly TALE‐dependent and could be activated by several different Xcc TALE proteins. Evaluation of Xcc strains from different continents showed that the Xcc‐TALE‐trap mediates resistance to this global panel of Xcc isolates. We also studied in planta‐evolved TALEs (eTALEs) with novel DNA‐binding domains and found that these eTALEs also activate the Xcc‐TALE‐trap, suggesting that the Xcc‐TALE‐trap is likely to confer durable resistance to Xcc. Finally, we show that the Xcc‐TALE‐trap confers resistance not only in laboratory infection assays but also in more agriculturally relevant field studies. In conclusion, transgenic plants containing the Xcc‐TALE‐trap offer a promising sustainable approach to control CBC. [ABSTRACT FROM AUTHOR]

Several studies have shown that spray-induced gene silencing (SIGS) in which double-stranded RNAs (dsRNAs) and small RNAs duplexes (sRNAs) are sprayed on plant surfaces to target pathogens genes or plant genes causing selective knockdown and provide effective crop protection. However, using SIGS to control bacterial diseases in plants has not been developed because the silencing phenomenon known as RNAi (RNA interference) is not present in bacteria. Here, we mainly focus on the applicability to use SIGS as an alternative eco-friendly strategy to control citrus canker caused by Xanthomonas; in parallel with citrus leaf miner control as a part of RNA-based citrus canker controlling strategy. We suggest several promising genes to be targeted or knocked down in citrus, citrus leaf miner and Xanthomonas, in addition to the application strategies of RNA molecules to enhance their uptake. Since the molecular mechanism of SIGS may be controlled by RNAi-like silencing machinery in Xanthomonas, it is important to study SIGS pathway components to better understand Xanthomonas virulence. Therefore, we propose a hypothetical and possible SIGS pathway; but this should be confirmed experimentally to insure the successful application of SIGS against citrus canker and other bacterial diseases. [ABSTRACT FROM AUTHOR]

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