Xanthomonas Arboricola Pv Research Articles

In vitro and in vivo antimicrobial activity of the fungal metabolite toluquinol against phytopathogenic bacteria.

Bacterial plant diseases cause tremendous economic losses worldwide. However, a few effective and sustainable control methods are currently available. To discover novel and effective management approaches, we screened marine fungi for their antibacterial activity against phytopathogenic bacteria in vitro and in vivo. We screened the culture broth of 55 fungal strains isolated from various marine sources (seawater, algae, and sediment) for their in vitro antibacterial activity using the broth microdilution method. Then, only the fungal strain (designated UL-Ce9) with higher antibacterial activity in vitro was tested in an in vivo experiment against tomato bacterial wilt. The active compounds of UL-Ce9 were extracted using ethyl acetate, purified by a series of chromatography, and the structure was elucidated by nuclear magnetic resonance spectroscopy. Pesticide formulations of toluquinol were prepared as soluble concentrates and wettable powder. The disease control efficacy of toluquinol formulations was evaluated against blight of rice and the bacterial wilt of tomato. The culture broth of UL-Ce9 showed high antibacterial activity against Agrobacterium tumefaciens, Ralstonia solanacearum, and Xanthomonas arboricola pv. pruni in vitro, and we selected UL-Ce9 for the in vivo test. The UL-Ce9 culture broth completely suppressed the bacterial wilt of tomato at a dilution of 1:5. The phylogenetic analysis identified UL-Ce9 as Penicillium griseofulvum, and the antibacterial metabolites were revealed as patulin, gentisyl alcohol, and toluquinol, all of which were associated with the biosynthetic pathway of the mycotoxin patulin. Patulin exhibited the highest antibacterial activity against 16 phytopathogenic bacteria in vitro, followed by toluquinol and gentisyl alcohol. As patulin is toxic, we selected toluquinol to investigate its potential use as a pesticide against bacterial plant diseases. Compared with the chemicals currently being applied in agriculture (streptomycin and oxytetracycline), toluquinol formulations exhibited similar and higher control efficacies against bacterial leaf blight of rice and bacterial wilt of tomato, respectively. To the best of our knowledge, this is the first report of the antibacterial activity of toluquinol against phytopathogenic bacteria. Our results suggest that toluquinol is a potential candidate for the development of novel and effective pesticides for the management of bacterial plant diseases.

Effects of Leaf Wetness Duration, Temperature, and Host Phenological Stage on Infection of Walnut by Xanthomonas arboricola pv. juglandis.

Bacterial blight, caused by Xanthomonas arboricola pv. juglandis, is a significant disease affecting walnut production worldwide. Outbreaks are most severe in spring, and closely tied to host phenology and weather conditions. Pathogen infections are mainly observed in catkins, female flowers, leaves, and fruits. In this study, the effect of wetness duration and temperature on walnut infections by X. arboricola pv. juglandis was determined through two independent experiments conducted under controlled environmental conditions. The combined effect of both climatic parameters on disease severity was quantified using a third-order polynomial equation. The model obtained by linear regression and backward elimination technique fitted well to the data (R2 = 0.94 and R2adj = 0.93). The predictive capacity of the forecasting model was evaluated on pathogen-inoculated walnut plants exposed to different wetness duration-temperature combinations under Mediterranean field conditions. Observed disease severity in all events aligned with predicted infection risk. Additionally, the relationship between leaf and fruit age and the disease severity was quantified and modelled. A prediction model, which has been referred to as the WalBlight-risk model, is proposed for evaluation as an advisory system for timing bactericide sprays to manage bacterial blight in Mediterranean walnut orchards.

Functional analysis of 3-Oxacyl-Acyl carrier protein reductase homolog (FabG3) in Xanthomonas arboricola pv. juglandis

Most of the pathogenic microorganisms that cause serious production and economic losses for the agricultural industry are members of the genus Xanthomonas. The main bacterial pathogen causing walnut black spot is Xanthomonas arboricola pv. juglandis (Xaj). Members of the genus Xanthomonas produce a yellow pigment called xanthomonadin, which is membrane-bound and protects the bacteria against photobiological and peroxidative damage. Here, we report the characterization of a gene named XajfabG3 (a homologous gene of fabG3 in Xanthomonas campestris pv. campestris), demonstrating that it is responsible for xanthomonadin synthesis. Expression of XajfabG3 in Escherichia coli fabG temperature-sensitive mutant CL104 restored the growth of CL104 under nonpermissive temperature, although XajFabG3 had a lower activity than XajFabG1 or FabG from Escherichia coli. In vitro assays demonstrated that XajFabG3 could reduce 3-oxoacyl-acyl carrier proteins (-ACPs) of fatty acid synthesis reactions and showed that although deletion of XajfabG3 did not affect bacterial growth and fatty acid composition, xanthomonadin production was abolished in a XajfabG3-deficient strain. Xanthomonadin production could be restored only by wild-type fabG3 and not by XajfabG1, another 3-oxoacyl-ACP reductase-encoding gene in Xaj, indicating that XajFabG3 specifically participates in the biosynthesis of xanthomonadin. Moreover, our study showed that the XajfabG3 knockout mutant affected virulence in the host Juglans regia L. Our elucidation of the biological function of the fabG3 gene in Xaj provides a target for the development of prevention and treatment agents for walnut blight.

Antibacterial effect of essential oils and their components against Xanthomonas arboricola pv. pruni revealed by microdilution and direct bioautographic assays.

Bacterial spot of stone fruits caused by Xanthomonas arboricola pv. pruni (Xap) is one of the most significant diseases of several Prunus species. Disease outbreaks can result in severe economic losses while the control options are limited. Antibacterial efficacy of essential oils (EOs) of thyme, cinnamon, clove, rosemary, tea tree, eucalyptus, lemon grass, citronella grass, and lemon balm was assessed against two Hungarian Xap isolates. The minimal inhibitory concentration (MIC) was determined by broth microdilution assay and for the identification of active EOs' components a newly introduced high-performance thin-layer chromatography (HPTLC)-Xap (direct bioautography) method combined with solid-phase microextraction-gas chromatography/mass spectrometry (SPME-GC/MS) was applied. All EOs inhibited both bacterium isolates, but cinnamon proved to be the most effective EO with MIC values of 31.25 µg/mL and 62.5 µg/mL, respectively. Compounds in the antibacterial HPTLC zones were identified as thymol in thyme, trans-cinnamaldehyde in cinnamon, eugenol in clove, borneol in rosemary, terpinen-4-ol in tea tree, citral (neral and geranial) in lemon grass and lemon balm, and citronellal and nerol in citronella grass. Regarding active compounds, thymol had the highest efficiency with a MIC value of 50 µg/mL. Antibacterial effects of EOs have already been proven for several Xanthomonas species, but to our knowledge, the studied EOs, except for lemon grass and eucalyptus, were tested for the first time against Xap. Furthermore, in case of Xap, this is the first report demonstrating that direct bioautography is a fast and suitable method for screening anti-Xap components of complex matrices, like EOs.

First Report of the Occurrence of the Bacterial Spot Disease of Stone Fruits Caused by Xanthomonas arboricola pv. pruni on Almond in Greece.

In 2021, two samples of almond (Prunus dulcis (Mill) Webb) shoots with symptoms resembling those caused by Xanthomonas arboricola pv. pruni (Xap), were examined at the Benaki Phytopathological Institute. The first sample was collected in June from a 0.4-ha orchard of fifteen-year-old almond trees (cv. 'Texas') with 40% disease incidence, in the Regional Unit of Serres (Northern Greece). Leaves exhibited, mainly at their tip and margins, small, angular, necrotic spots with chlorotic halo, often coalesced into larger necrotic lesions which fell out leaving leaves with a 'shot-hole' like appearance. Fruits displayed dark brown, sunken, corky, gum oozing lesions and shoots developed dark brown, elongated, slightly sunken lesions. Bacterial streaming from the marginal areas of necrotic lesions was observed microscopically. On the lesions of fruits, leaves and shoots, Xap was detected by immunofluorescence assay (IF) using polyclonal antibodies (Plant Research International, the Netherlands) and two qPCR assays (Garita-Cambronero et al. 2017; Palacio-Bielsa et al. 2011). Eight Xanthomonas-like isolates obtained on the SP agar (Hayward 1960) and Nutrient agar (Schaad et al. 2001) culture media were Gram-negative, oxidase negative, strictly aerobic, sensitive to 0.1% w/v TTC, hydrolysing gelatin and Tween 80 but not starch, and also inducing hypersensitive response in tomato plants, as expected for Xap (Schaad et al. 2001). Isolates' identification was confirmed by the IF and the two qPCR assays cited above, as well as a conventional PCR (Pothier et al., 2011). Infiltration of a suspension (107 cfu/ml) of one isolate into five leaves of a two-year-old almond tree cv. 'Texas', and also into five detached leaves from the same tree (Randhawa and Civerolo 1985), caused necrotic spots on all inoculated leaves (10 inoculation sites/leaf), after a four day incubation period at 25oC under high humidity. The Xap reference strain NCPPB 3877 and sterile water were used as positive and negative controls, respectively. The pathogen was reisolated from necrotic spots of the inoculated leaves and identified by IF and two qPCR assays, as previously. The second sample was collected by a grower in September from a 3.7-ha orchard of five-year-old almond trees (cv. 'Tuono') exhibiting 50% disease incidence, in the Regional Unit of Fthiotida (Central Greece). Leaves and fruits showed symptoms similar to those described for the first sample, except that, lesions on fruits, which were at a stage of advanced mesocarp dehydration, were raised. Five Xap isolates were obtained from symptomatic leaves and fruits, and their pathogenicity on almond was confirmed, as in the first sample. Furthermore, sequences of PCR products using primers targeting the 16S-rDNA (Lane 1991;Lane et al., 1985), gyrB (Parkinson et al. 2007) and ftsX (Pothier et al. 2011) genes of two Xap isolates, one from fruit- and one from leaf-necrotic lesions of the first sample, were searched against the NCBI GenBank database, revealing that the obtained sequences of 16S-rRNA (OP412487; OP412488), gyrB (OP467593; OP467594) and ftsX (OP467595; OP467596) genes were 100% identical to the corresponding genomic regions of the Xap strains IVIA 2626.1 (CP076628.1) and CITA 33 (CP076701.1). This is the first report on the presence of Xap in Greece. As these Xap outbreaks have occurred in regions with extensive almond cultivation, a crop of great economic importance for Greece, measures for its eradication have already been advised.

Population characteristics of Xanthomonas arboricola pv. corylina strains from hazelnut orchards in Turkey

Population characteristics of Xanthomonas arboricola pv. corylina strains from hazelnut orchards in Turkey

Protocatechuic acid, ferulic acid and relevant defense enzymes correlate closely with walnut resistance to Xanthomonas arboricola pv. juglandis

BackgroundJuglans regia L. is an important nut tree that has a wide range of distribution in temperate regions of the world. In some walnut orchards, walnut blight can become a problematic disease that affects the growth of walnut trees. To explore the correlation between biochemical response and walnut resistance, we inoculated four walnut cultivars with Xanthomonas arboricola pv. juglandis (Xaj). The walnut cultivars were, namely, ‘Xiangling’, ‘Xiluo 2’, ‘Yuanfeng’ and ‘Xifu 2’. Total phenol content (TPC) and total flavonoid content (TFC) were measured, whereby nine major phenolic compounds and several relevant enzymes were identified.ResultsThe results showed that the most resistant and susceptible walnut varieties were ‘Xiluo 2’ and ‘Xifu 2’ respectively. The reaction of walnut to Xaj was characterized by the early accumulation of phenolic compounds in the infected site. After inoculation with Xaj, we found that the resistant variety ‘Xiluo 2’ show the significant differences with other varieties at different time points through the determination of related antioxidant enzymes such as catalase (CAT) and peroxidase (POD). Meanwhile, the phenylalanine ammonia lyase (PAL) of ‘Xiluo 2’ increased significantly at 8 day post infection (dpi) and made differences from the control samples, while other varieties changed little. And the polyphenol oxidase (PPO) was significantly higher than in the control at 16 dpi, maintaining the highest and the lowest activity in ‘Xiluo 2’ and ‘Xifu 2’ respectively. It was also found that the content of protocatechuic acid in all cultivars increased significantly at 4 dpi, and ‘Xiluo 2’ was significantly higher than that of the control. In the early stage of the disease, ferulic acid content increased significantly in ‘Xiluo 2’.ConclusionOur findings confirmed that the metabolism of phenolic compounds and related defense enzymes are of great significance in the response of walnut to Xaj.

Hazelnut-Associated Bacteria and Their Implications in Crop Management

In recent years, the cultivation of hazelnut (Corylus avellana) has expanded in several areas of Europe, Asia, Africa, and North and South America following the increased demand for raw materials by the food industry. Bacterial diseases caused by Xanthomonas arboricola pv. corylina and Pseudomonas avellanae are threats of major concern for hazelnut farmers. These pathogens have been controlled with copper-based products, which are currently being phased out in the European Union. Following the need for alternative practices to manage these diseases, some progress has been recently achieved through the exploitation of the plant’s systemic acquired resistance mechanisms, nanoparticle technology, as well as preventive measures based on hot water treatment of the propagation material. However, bacteria are not only agents of the biotic adversities of hazelnut. In fact, the application of plant growth-promoting rhizobacteria at the seedling level could enhance better performance of the tree. Likewise, endophytic and epiphytic microorganisms are considered to play a notable role in plant nutrition and protection, and their effects on hazelnut fitness deserve to be further investigated. Finally, bacterial associations may also be relevant in the post-harvest phase, particularly with reference to the processes of lipid oxidation and fat degradation suffered by the kernels after grinding.

Short Communication: Genetic diversity of walnut blight-associated bacteria from China using rep-PCR

Abstract. Zheng C, Zhu J, Yang X, Fu B. 2022. Short Communication: Genetic diversity of walnut blight-associated bacteria from China using rep-PCR. Biodiversitas 23: 5681-5686. Walnut blight is caused by the bacterium Xanthomonas arboricola pv. juglandis (Xaj), is one of the most serious diseases in walnut production around the world. Understanding the genetic diversity of walnut blight pathogen is of great significance to developing a scientific and reasonable prevention and control strategy. The rep-PCR (Repetitive Sequence Polymerase Chain Reaction) technique is a powerful tool for analyzing plant pathogens. In this paper, using three pairs of rep-PCR universal primers (ERIC, BOX, and REP), a genetic diversity analysis was carried out on walnut blight-associated bacteria mainly isolated from different counties of Hubei Province, China. A study of 60 walnut blight-associated bacteria isolated in 2016 found that they had a high genetic similarity among them. There is no clear correlation between the genetic diversity of these isolates and their geographical or tissue origins. Interestingly, these clusters can also distinguish copper-resistant isolates from sensitive ones. Therefore, rep-PCR is a powerful tool to study the population of walnut pathogenic bacteria.

Polyphasic study of phytopathogenic bacterial strains associated with deep bark canker of walnut in Serbia revealed a new species, Brenneria izbisi sp. nov.

Serious outbreaks of walnut deep bark canker were observed on young walnut trees (Juglans regia L.) in two localities in the northern part of Serbia during 2020. From the symptomatic walnut tissues, two types of bacterial colonies were isolated, predominantly, light cream, circular and smooth colonies, as well as small, yellowish, mucoid and convex ones. PCR analysis and phenotypic assays suggested that the former group belongs to Brenneria spp., while the latter isolates were identified as Xanthomonas arboricola pv. juglandis. Within the Brenneria group, two strains were identified as Brenneria nigrifluens, while other 15 strains did not belong to any Brenneria species described so far. Therefore, we selected four representative strains of the unknown Brenneria sp. and subjected them to polyphasic analysis. As expected, in a phylogenetic tree based on partial 16S rDNA sequences, four novel strains grouped with other Brenneria representatives, and showed close phylogenetic relationship to Brenneria salicis. Furthermore, multilocus sequence analysis (MLSA) based on the partial sequences of atpD, gyrB, infB and rpoB housekeeping genes and core-genome phylogeny indicated that the studied strains form a novel and a clearly separate Brenneria lineage. Overall genome relatedness indices showed that they represent a new Brenneria species. The new species can be differentiated from the other Brenneria spp. infecting walnut and closely related B. salicis strains based on phenotypic characteristics, as well. Moreover, the pathogenicity tests on two-year-old walnut plants proved the ability of strains to cause necrosis and longitudinal black lesions and cracks on the trunk and branches of walnut trees. Overall, polyphasic characterization showed that the studied strains isolated from walnut with symptoms of deep bark canker represent a novel species of the genus Brenneria for which the name Brenneria izbisi sp. nov. is proposed. The type strain of B. izbisi is KBI 423T (= CFBP 9035T = LMG 32479T). To facilitate rapid identification of newly described species, a conventional PCR protocol and primers targeting the putative gene hrpP, were developed. Further study should reveal the potential role of each pathogen isolated from symptomatic walnut in disease development as well as possible interaction between them.

Complete Genome Sequence of Xanthomonas arboricola pv. pruni Strain Xcp1 Isolated in 1984 from a Bacterial Spot Spring Canker on Prunus persica var. nucipersica cv. "Redgold".

Xanthomonas arboricola pv. pruni is an important plant pathogen and the causal agent of bacterial spot of stone fruits (Prunus spp). Here, we report a complete genome of X. arboricola pv. pruni strain Xcp1 generated from hybrid PacBio Sequel and Illumina NextSeq2000 sequencing.

Comparative Analysis of Novel Lytic Phages for Biological Control of Phytopathogenic Xanthomonas spp.

Xanthomonas is an important genus of plant-pathogenic bacteria that affects agronomic and economically important crops, causing serious economic losses. In fact, several Xanthomonas species are considered regulated quarantine pests. Due to the lack of effective control measures to treat plant-pathogenic bacteria, innovative control tools are needed to carry out integrated disease management. In this regard, bacteriophages (phages), viruses of bacteria, constitute a promising biocontrol tool. In this work, we report the isolation and characterization of 11 novel Xanthomonas arboricola pv. juglandis phages belonging to different families and genera of the class Caudoviricetes. Infectivity matrix in more than 60 isolates of different xanthomonads and other phytopathogenic bacteria suggests that these phages are specific to the Xanthomonas genus, with different host ranges depending on the isolates tested. Interestingly, some of these phages showed relevant features to be used as biocontrol tools to combat pathogenic Xanthomonas spp. as important as X. oryzae or X. citri. IMPORTANCE Phytopathogenic bacteria represent serious losses worldwide. The lack of current treatments has focused the spotlight on phages, viruses of bacteria, as very promising biocontrol tools. Phages are very specific and can help to control bacterial infections in crops, as is the case of xanthomonads-associated diseases. The discovery of new environmental phages with lytic capacity that can help to combat these pathogens is of special relevance, and it is necessary to implement phage isolation and characterization techniques to determine their host range and their genomic properties. The establishment of phage collections worldwide will allow their use as preventive, diagnostic, or therapeutic tools. Although there is still a long way to go, this work is a step forward in the implementation of new ecofriendly techniques to combat key pathogens in the field.

Characterization of Xanthomonas arboricola pv. pruni from Prunus spp. orchards in Western Balkans

AbstractThe present study provides a new insight into the existing Xanthomonas arboricola pv. pruni (Xap) bacterial population originating from peach and apricot in two Western Balkans countries (Serbia and Montenegro). Multilocus sequence typing and analysis of the sequences of nine housekeeping genes revealed homology between the tested Xap strains as well as with the European population of this bacterium. The tested strains share the same haplotype (Haplotype I) with the Xap strains from Italy, France, Spain, United States, Australia and Brazil. The revealed single nucleotide change (G ↔ C) in the sequences of the gyrB1 gene differentiates Haplotype I from Haplotype II (Xap from South Korea, New Zealand, Argentina and Uruguay). The detached‐leaf bioassay results confirmed differences in virulence between strains originating from peach and apricot towards Prunus armeniaca (apricot), indicating host specialization of the apricot strain towards this host. For the first time, immunity of P. fruticosa (European ground cherry) to Xap was established. According to the AUDPC, PCA and cluster analysis, other Prunus spp. were classified as having low susceptibility (P. mahaleb, P. cerasus and P. avium), as susceptible (P. domestica) and as highly susceptible (P. persica, P. dulcis, P. cerasifera and P. spinosa). Xap strains were also found to be susceptible to 10 tested antibiotics. This study provides valuable knowledge on the Xap population from stone fruit grown in the Western Balkans region as well as the source of immunity, which could serve as a starting point for breeding Prunus cultivars and could be used as the main control strategy.

Identification, Genetic Diversity, and Chemical Control of Xanthomonas arboricola pv. pruni in China.

Peach bacterial spot caused by Xanthomonas arboricola pv. pruni has become widespread in most peach-producing areas of China and has caused devastating losses to the peach industry. However, little is known about the population biology and epidemiology of X. arboricola pv. pruni in China, thus no effective management strategy is available. Altogether, 321 symptomatic samples of peach bacterial spot from 12 provinces in China were collected from which 612 bacterial isolates were obtained. Based on 16S rDNA sequence comparison in GenBank, the obtained isolates were identified as Pantoea spp. (514) and Xanthomonas spp. (98). The pathogenicity test demonstrated that the causal agent of the peach bacterial spot was the Xanthomonas spp. instead of the Pantoea spp. Based on morphological observation, physiological and biochemical characterization, and molecular identification, the Xanthomonas spp. were further identified to be X. arboricola pv. pruni. Then, 41 X. arboricola pv. pruni isolates representing different populations were selected and analyzed with repetitive element sequence based-PCR and intersimple sequence repeat markers to understand the genetic diversity and population structure along with four X. arboricola pv. pruni isolates from plum and three isolates of X. arboricola pv. juglandis as comparison. A total of 98 polymorphic alleles were identified, with a mean value of percentage of polymorphic loci of 14. Genetic diversity and phylogenetic analysis revealed the profound heterogeneity between X. arboricola pv. juglandis and X. arboricola pv. pruni, moderate genetic differentiation within X. arboricola pv. pruni, and obvious host specificity but weak geographical differentiation in X. arboricola population. Finally, the efficiency of bactericides on X. arboricola pv. pruni was evaluated invitro and invivo. The parallel repeated field trials in two orchards demonstrated that 80% Mancozeb (1:800) and 47% Kocide (1:800, 1:1,500, and 1:2,000) had excellent control efficacies for X. arboricola pv. pruni, especially as the control efficacy of Kocide could even reach 90%. This study conducted a systematic investigation for the occurrence, population variance, and chemical control of X. arboricola pv. pruni. It improved the understanding of the pathogen populations of peach bacterial spot in China and provided solid theoretical and practical guidance for X. arboricola pv. pruni control.

Characterization of Xanthomonas arboricola pv. juglandis Bacteriophages against Bacterial Walnut Blight and Field Evaluation.

Xanthomonas arboricola pv. juglandis (hereafter X. juglandis) is the etiological agent of walnut blight, the most important bacterial disease affecting walnut production worldwide. Currently, the disease is treated mainly with copper-derived compounds (e.g., CuSO4) despite the evidence of genetic resistance in these strains. Regarding the effectiveness and sustainability, the use of a bacteriophage appears to be a biocontrol alternative to reduce X. juglandis load and symptomatology of walnut blight. Here, the phages f20-Xaj, f29-Xaj, and f30-Xaj were characterized, and their effectiveness in walnut orchards against walnut blight was determined. These bacteriophages showed a specific lytic infection in X. juglandis strains isolated from Chile and France. Phylogenetic analysis of the complete genome of f20-Xaj and f30-Xaj indicates that these phages belong to the Pradovirus genus. In the field, the cocktail of these bacteriophages showed similar effectivity to CuSO4 in the reduction of incidence and severity in walnut tissue. Moreover, the bacterial load of X. juglandis was significantly reduced in the presence of bacteriophages in contrast to a CuSO4 treatment. These results show that the use of bacteriophages can be an alternative to combat the symptoms of walnut blight caused by X. juglandis.

Commodity risk assessment of Prunus domestica plants from Ukraine.

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as ‘High risk plants, plant products and other objects’. This Scientific Opinion covers plant health risks posed by plants of Prunus domestica grafted on Prunus cerasifera imported from Ukraine, taking into account the available scientific information, including the technical information provided by Ukraine. All pests associated with the commodity were evaluated against specific criteria for their relevance for this opinion. One quarantine pest (Lopholeucaspis japonica), two protected zone quarantine pests (Erwinia amylovora and Xanthomonas arboricola pv. pruni) and one non‐regulated pest (Eotetranychus prunicola) that fulfilled all relevant criteria were selected for further evaluation. For these four pests, the risk mitigation measures proposed in the technical dossier from Ukraine were evaluated taking into account the possible limiting factors. For the selected pests, an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. The degree of pest freedom varies among the pests evaluated, with Xanthomonas arboricola pv. pruni being the pest most frequently expected on the imported plants. The Expert Knowledge Elicitation indicated with 95% certainty that between 9,870 and 10,000 bundles (consisting of 10 plants each) per 10,000 would be free from Xanthomonas arboricola pv. pruni.

Peptide Conjugates Derived from flg15, Pep13, and PIP1 That Are Active against Plant-Pathogenic Bacteria and Trigger Plant Defense Responses.

ABSTRACTThirty peptide conjugates were designed by combining an antimicrobial peptide (BP16, BP100, BP143, KSL-W, BP387, or BP475) at the N- or C-terminus of a plant defense elicitor peptide (flg15, BP13, Pep13, or PIP1). These conjugates were highly active in vitro against six plant-pathogenic bacteria, especially against Xanthomonas arboricola pv. pruni, Xanthomonas fragariae and Xanthomonas axonopodis pv. vesicatoria. The most active peptides were those incorporating Pep13. The order of the conjugation influenced the antibacterial activity and the hemolysis. Regarding the former, peptide conjugates incorporating the elicitor peptide flg15 or Pep13 at the C-terminus were, in general, more active against Pseudomonas syringae pv. actinidiae and P. syringae pv. syringae, whereas those bearing these elicitor peptides at the N-terminus displayed higher activity against Erwinia. amylovora and the Xanthomonas species. The best peptide conjugates displayed MIC values between 0.8 and 12.5 μM against all the bacteria tested and also had low levels of hemolysis and low phytotoxicity. Analysis of the structural and physicochemical parameters revealed that a positive charge ranging from +5 to +7 and a moderate hydrophobic moment/amphipathic character is required for an optimal biological profile. Interestingly, flg15-BP475 exhibited a dual activity, causing the upregulation of the same genes as flg15 and reducing the severity of bacterial spot in tomato plants with a similar or even higher efficacy than copper oxychloride. Characterization by nuclear magnetic resonance (NMR) of the secondary structure of flg15-BP475 showed that residues 10 to 25 fold into an α-helix. This study establishes trends to design new bifunctional peptides useful against plant diseases caused by plant-pathogenic bacteria.IMPORTANCE The consequences of plant pathogens on crop production together with the lack of effective and environmentally friendly pesticides evidence the need of new agents to control plant diseases. Antimicrobial and plant defense elicitor peptides have emerged as good candidates to tackle this problem. This study focused on combining these two types of peptides into a single conjugate with the aim to potentiate the activity of the individual fragments. Differences in the biological activity of the resulting peptide conjugates were obtained depending on their charge, amphipathicity, and hydrophobicity, as well as on the order of the conjugation of the monomers. This work provided bifunctional peptide conjugates able to inhibit several plant-pathogenic bacteria, to stimulate plant defense responses, and to reduce the severity of bacterial spot in tomato plants. Thus, this study could serve as the basis for the development of new antibacterial/plant defense elicitor peptides to control bacterial plant pathogens.

De Novo Arginine Synthesis Is Required for Full Virulence of Xanthomonas arboricola pv. juglandis During Walnut Bacterial Blight Disease.

Walnut blight (WB) disease caused by Xanthomonas arboricola pv. juglandis (Xaj) threatens orchards worldwide. Nitrogen metabolism in this bacterial pathogen is dependent on arginine, a nitrogen-enriched amino acid that can either be synthesized or provided by the plant host. The arginine biosynthetic pathway uses argininosuccinate synthase (argG), associated with increased bacterial virulence. We examined the effects of bacterial arginine and nitrogen metabolism on the plant response during WB by proteomic analysis of the mutant strain Xaj argG-. Phenotypically, the mutant strain produced 42% fewer symptoms and survived in the plant tissue with 2.5-fold reduced growth compared with wild type, while showing itself to be auxotrophic for arginine invitro. Proteomic analysis of infected tissue enabled the profiling of 676 Xaj proteins and 3,296 walnut proteins using isobaric labeling in a data-dependent acquisition approach. Comparative analysis of differentially expressed proteins revealed distinct plant responses. Xaj wild type (WT) triggered processes of catabolism and oxidative stress in the host under observed disease symptoms, while most of the host biosynthetic processes triggered by Xaj WT were inhibited during Xaj argG- infection. Overall, the Xaj proteins revealed a drastic shift in carbon and energy management induced by disruption of nitrogen metabolism while the top differentially expressed proteins included a Fis transcriptional regulator and a peptidyl-prolyl isomerase. Our results show the critical role of de novo arginine biosynthesis to sustain virulence and minimal growth during WB. This study is timely and critical as copper-based control methods are losing their effectiveness, and new sustainable methods are urgently needed in orchard environments.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Copper Tolerance in Xanthomonas arboricola pv. pruni in South Carolina Peach Orchards.

Bacterial spot of peach, caused by Xanthomonas arboricola pv. pruni, causes yield loss every year in southeastern U.S. peach orchards. Management is mainly driven by season-long applications of copper-based products, site location, and choice of cultivar. Although tolerance to copper has not been reported in X. arboricola pv. pruni in the United States, adaptation of populations from frequent use is a concern. We collected X. arboricola pv. pruni from shoot cankers, leaves, and fruit of cultivar O'Henry over 2 years from three conventional farms and one organic farm in South Carolina, one orchard per farm. The four farms had been using copper extensively for years to control bacterial spot. X. arboricola pv. pruni was isolated from four canker types (bud canker, tip canker, nonconcentric canker, and concentric canker) in early spring (bud break), as well as from leaf and fruit tissues later in the season at the phenological stages of pit hardening and final swell. X. arboricola pv. pruni was most frequently isolated from cankers of the organic farm (24% of the cankers) and most isolates (45%) came from bud cankers. X. arboricola pv. pruni isolates were assessed for sensitivity to copper using minimal glucose yeast agar and nutrient agar amended with 38 μg/ml or 51 μg/ml of Cu2+. Two phenotypes of copper tolerance in X. arboricola pv. pruni were discovered: low copper tolerance (LCT; growth up to 38 μg/ml Cu2+) and high copper tolerance (HCT; growth up to 51 μg/ml Cu2+). A total of 26 (23 LCT and 3 HCT) out of 165 isolates in 2018 and 32 (20 LCT and 12 HCT) out of 133 isolates in 2019 were tolerant to copper. Peach leaves on potted trees were sprayed with copper rates typically applied at the stages of delayed dormancy (high rate; 2,397 μg/ml Cu2+), shuck split (medium rate; 599 μg/ml Cu2+), and during summer cover sprays (low rate; 120 μg/ml Cu2+), and subsequently inoculated with sensitive, LCT, and HCT strains. Results indicated that the low and medium rates of copper reduced bacterial spot incidence caused by the sensitive strain but not by the LCT and HCT strains. This study confirms existence of X. arboricola pv. pruni tolerance to copper in commercial peach orchards in the southeastern United States, and suggests its contribution to bacterial spot development under current management practices.

Complete Genome and Plasmid Sequence Data of Three Strains of Xanthomonas arboricola pv. corylina, the Bacterium Responsible for Bacterial Blight of Hazelnut.

Xanthomonas arboricola pv. corylina is the causal agent of bacterial blight of hazelnut. The bacterium has been listed as an A2 quarantine pathogen in Europe since 1978 and on the regulated non-quarantine pest list since 2019. Three isolates from various geographic regions and isolated at different times were sequenced using a hybrid approach with short- and long-read technologies to generate closed genome and plasmid sequences in order to better understand the biology of this pathogen.