Canker Lesions Research Articles

Deciphering how plant pathogenic bacteria disperse and meet: Molecular epidemiology of Xanthomonas citri pv.citri at microgeographic scales in a tropical area of Asiatic citrus canker endemicity.

Although some plant pathogenic bacteria represent a significant threat to agriculture, the determinants of their ecological success and evolutionary potential are still poorly understood. Refining our understanding of bacterial strain circulation at small spatial scales and the biological significance and evolutionary consequences of co‐infections are key questions. The study of bacterial population biology can be challenging, because it requires high‐resolution markers that can be genotyped with a high throughput. Here, we overcame this difficulty for Xanthomonas citri pv. citri, a genetically monomorphic bacterium causing Asiatic citrus canker (ACC). Using a genotyping method that did not require cultivating the bacterium or purifying DNA, we deciphered the pathogen's spatial genetic structure at several microgeographic scales, down to single lesion, in a situation of ACC endemicity. In a grove where copper was recurrently applied for ACC management, copper‐susceptible and copper‐resistant X. citri pv. citri coexisted and the bacterial population structured as three genetic clusters, suggesting a polyclonal contamination. The range of spatial dependency, estimated for the two largest clusters, was four times greater for the cluster predominantly composed of copper‐resistant bacteria. Consistently, the evenness value calculated for this cluster was indicative of increased transmission. Linkage disequilibrium was high even at a tree scale, probably due to a combination of clonality and admixture. Approximately 1% of samples exhibited within‐lesion multilocus polymorphism, explained at least in part by polyclonal infections. Canker lesions, which are of major biological significance as an inoculum source, may also represent a preferred niche for horizontal gene transfer. This study points out the potential of genotyping data for estimating the range of spatial dependency of plant bacterial pathogens, an important parameter for guiding disease management strategies.

First Report of Seimatosporium vitis Associated with Grapevine Trunk Diseases on Vitis vinifera in Italy

HomePlant DiseaseVol. 103, No. 4First Report of Seimatosporium vitis Associated with Grapevine Trunk Diseases on Vitis vinifera in Italy PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Seimatosporium vitis Associated with Grapevine Trunk Diseases on Vitis vinifera in ItalyI. Camele and S. M. MangI. Camele†Corresponding author: I. Camele; E-mail: E-mail Address: ippolito.camele@unibas.ithttp://orcid.org/0000-0001-8099-8234School of Agricultural, Forestry, Food and Environmental Sciences, University of Basilicata, 85100 Potenza, ItalySearch for more papers by this author and S. M. MangSchool of Agricultural, Forestry, Food and Environmental Sciences, University of Basilicata, 85100 Potenza, ItalySearch for more papers by this authorAffiliationsAuthors and Affiliations I. Camele † S. M. Mang School of Agricultural, Forestry, Food and Environmental Sciences, University of Basilicata, 85100 Potenza, Italy Published Online:13 Feb 2019https://doi.org/10.1094/PDIS-09-18-1686-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In spring 2018, in a vineyard (7 ha) located in Matera province (southern Italy), during a survey to verify the sanitary status of grapevines in Basilicata Region, Vitis vinifera plants (25-year-old, cv. Sangiovese) were found with typical “esca disease” symptoms on the leaves (tiger stripes). In addition, trunk cankers and internal wood necrosis were observed. More than 30% of the living plants were symptomatic and 10% more were dead. To isolate the likely pathogen, small pieces from trunk discs of living, symptomatic plants were surface sterilized with 70% alcohol, plated in Petri dishes containing potato dextrose agar (PDA), and incubated at 24°C for 14 days in darkness. From all samples, only a single fungal species was isolated, and this was identified as Seimatosporium vitis X.P. Xiao, Camporesi E. & K.D. Hyde through morphological comparison (Senanayake et al. 2015) and a molecular analysis based on three genes including the rDNA-ITS region (ITS), β-tubulin-2 (TUB2), and translation elongation factor 1-α (TEF1-alpha). The sequences of four pure-culture isolates were deposited in EMBL GenBank under accession numbers LS991528 to LS991531 (ITS), LS997596 to LS997599 (TUB-2), and LS999502 to LS999505 (TEF1-alpha). All sequences showed 99 to 100% identity with authentic S. vitis sequences already present in the GenBank database under the following accession numbers: ITS (KY632661, KX555652, and KU721882), TUB-2 (KY706248, KY706254, and KY706257), and TEF1-alpha (KY06328, KY06331, and KY06332). A phylogenetic tree, based on combined ITS, TUB-2, and TEF1-alpha data, was obtained using MEGA version 6.0 with the maximum parsimony analyses revealing that all isolates fell in the S. vitis clade, confirming the species identification. Koch’s postulates were fulfilled using four S. vitis isolates inoculated on woody stems of 2-year-old potted cultivar Sangiovese. Shoots were surface sterilized with 70% alcohol and then wounded with a sterile scalpel. Discs (0.5-mm diameter) from 14-day-old agar cultures were applied to the wound and sealed with Parafilm, and then the plants were kept in a growth chamber at 24°C and 90% humidity and 16-h light. Sterile PDA discs were used as controls. Each isolate was inoculated onto 10 plants, and five others were used as controls (45 plants in total). The pathogenicity test was carried out twice. About 45 days after inoculation, each inoculated shoot presented a canker lesion about 3 to 4 cm long around the inoculation point and interveinal chlorosis on leaves. Control plants were always symptomless. Only S. vitis was consistently reisolated from inoculated tissues. Pathogenicity assays confirmed that S. vitis from V. vinifera plants is the causal agent of the observed symptoms in grapevine. S. vitis was reported to be associated with grapevine trunk diseases (GTDs) in Hungary (Váczy 2017) and in California (Lawrence et al. 2018). Senanayake et al. (2015) had earlier reported S. vitis from Italy but not in association with GTDs and without pathogenicity tests. This is the first report of S. vitis associated with the GTDs in Italy.

Formation of Citrus Canker Lesions

Citrus canker is a non-systemic bacterial disease that affects citrus trees in both the citrus nursery and in commercial plantings. This poster is designed to assist citrus nursery workers in the identification of citrus canker. This one-page document was written by Jamie Burrow, Megan Dewdney, Ajia Paolillo, and Tim Riley and published by the UF/IFAS Plant Pathology Department. http://edis.ifas.ufl.edu/pp346

Changes in the physico-chemical properties of the xanthan produced by Xanthomonas citri subsp. citri in grapefruit leaf extract.

Xanthan is a virulence factor produced by Xanthomonas spp. We previously demonstrated that this exopolysaccharide is not only essential for pathogenicity by contributing with bacterial survival but also its pyruvate substituents interfere with some plant defense responses. Deepening our studies about xanthan properties and structure, the aim of this work was to analyze the characteristics of xanthan produced by Xanthomonas in different culture media. We analyzed the xanthan produced by Xanthomonas citri subsp. citri (Xcc) in leaf extracts from grapefruit (a susceptible host of this bacterium) and compared it with the xanthan produced in a synthetic culture medium. We found that the xanthan produced in the grapefruit extract (Xan-GLE) presented shorter and more disordered molecules than xanthan produced in the synthetic medium (Xan-PYM). Besides, Xan-GLE resulted less viscous than Xan-PYM. The disordered molecular conformation of Xan-GLE could be attributed to its higher pyruvilation degree and lower acetylation degree compared with those detected in Xan-PYM. Meanwhile, the difference in the viscosity of both xanthans could be due to their molecules length. Finally, we cultured Xcc in the presence of the Xan-GLE or Xan-PYM and observed the formation of biofilm-like structures in both cases. We found significant differences in biofilm architecture between the two conditions, being the biofilm produced in presence of Xan-GLE similar to that formed in canker lesions developed in lemon plant leaves. Together, these results show how xanthan structure and properties changed when Xcc grew in a natural substrate and can contribute to better understand the biological role of xanthan.

Characteristics of Citrus Canker Lesions Associated with Premature Drop of Sweet Orange Fruit.

During the development of a citrus fruit, many cycles of infection by Xanthomonas citri subsp. citri may occur leading to the development of a range of characteristics of citrus canker lesions scattered across the fruit surface. This study aimed to determine whether the size of the lesions, their distance from the peduncle, and the number and time of appearance of the lesions on fruit of sweet orange were associated with premature fruit drop. A multiple linear regression analysis revealed a negative relationship between the fruit detachment force and the lesion diameter, the proximity of the nearest lesion to the peduncle and the number of lesions. A survival analysis demonstrated that these characteristics significantly influenced the probability and the time that a cankered fruit remained attached to the tree. More than 90% of dropped fruit had large lesions (>5 mm) but not all fruit with large lesions dropped before harvest. Approximately 50% of the harvested fruit had lesions >5 mm. On the harvested fruit remaining on the tree, although large, the lesions had a smaller diameter, were located farther from the peduncle, and were less numerous than those observed on dropped fruit. Small canker lesions neither reduced the detachment force nor the survival of fruit in the tree. The earlier a fruit expressed canker symptoms, the higher the probability the fruit developed large lesions near the peduncle and/or developed lesions in greater numbers. This study provides a better understanding on the relationship between the time of appearance of lesions of citrus canker on fruit and premature fruit drop. This information defines the critical period for fruit protection and may be used to improve disease management.

Bioguided isolation, characterization and media optimization for production of Lysolipins by actinomycete as antimicrobial compound against Xanthomonas citri subsp. citri.

Citrus Canker disease is one of the most important disease in citrus production worldwide caused by gram-negative bacterial pathogen Xanthomonas citri subsp. citri, leading to great economic losses. Currently, a spray of copper-based bactericides is the primary measure for citrus canker management. However, these measures can lead to the contamination of soil by metal contamination, but also the development of copper-resistant Xanthomonas populations. Considering the need to discovery new alternatives to control the citrus canker disease, actinomycetes isolated from the Brazilian Caatinga biome and their crude extracts were tested against different strains of Xanthomonas citri subsp. citri. Streptomyces sp. Caat 1-54 crude extract showed the highest antibiotic activity against Xcc. The crude extract dereplication was performed by LC-MS/MS. Through bioassay-guided fractionation strategy, the antimicrobial activity was assigned to Lysolipins, showing a MIC around 0.4-0.8µg/mL. Growth media optimization using statistical experimental design increased the Lysolipins production in three-fold production. The preventive and curative effects of the optimized crude extract obtained by experimental design of Caat-1-54 against citrus canker were evaluated in potted 'Pera' sweet orange nursery trees. Caat 1-54 extract was effective in preventing new infections by Xcc on leaves but was not able to reduce Xcc population in pre-established citrus canker lesions. Streptomyces sp. Caat 1-54 extract is a promising, environmentally-friendly source of antimicrobial compound to protect citrus trees against citrus canker.

First Report of Shoot Canker on Chestnut Caused by Diaporthe nobilis in Shandong Province of China.

Chinese chestnut (Castanea mollissima) is an economic tree widely cultivated for nut production. In May 2015, for the first time, canker and severe twig dieback were observed in the chestnut orchards in Yantai, Shandong Province, China. The cankers appeared on shoots, twigs, and branches with sunken reddish-brown/tan symptoms. The shoots died above the infected area when girdled. However, fruit rot was not observed. In later summer, numerous small black pycnidia were formed along the lenticels on the surface of diseased bark with curly slim and pale-yellow spore-tendrils being released from the pycnidia after rainfall. To identify the causal agents, tissue fragments (5 mm²) were excised from the canker lesions, and 10 isolates were obtained on potato dextrose agar (PDA) (Bai et al. 2015). These isolates formed creamy white mycelia with concentric rings on PDA. Black pycnidia developed in culture and produced two types of conidia: hyaline, fusiform-elliptic to oblong-elliptic alpha conidia and hyaline, filiform, slightly curved or hooked beta conidia. The size of alpha conidia varied from 7.5 to 12.5 × 2.1 to 2.8 μm, and that of beta conidia ranged from 26.8 to 30.2 × 1.2 to 1.7 μm. Based on morphological characteristics, these 10 isolates were close to Diaporthe nobilis CBS 587.79 (Gomes et al. 2013). To confirm the identification, DNA was extracted from the mycelium of three monoconidial isolates (CHNut1, CHNut2, and CHNut3). The internal transcribed spacer region (ITS), a partial sequence of calmodulin (CAL), histone H3 gene (HIS), translation elongation factor 1-α gene (TEF1), and β-tubulin gene (TUB) were amplified and sequenced using primers ITS1/ITS4, Bt2a/Bt2b, H3-1a/H3-1b (Glass and Donaldson 1995), EF1-728F/EF1-986R, and CAL-228F/CAL-737R (Carbone and Kohn 1999), respectively. The consensus sequences (GenBank accession nos. MG920017 for ITS, MG926648 for CAL, MG926649 for TEF1, MG926650 for TUB, and MG926651 for HIS) were aligned using BLASTn in GenBank showing high homology (99, 100, 99, 100, and 100%) with the sequences of ITS (KC343153), CAL (KC343395), TEF1 (KC343879), TUB (KC344121), and HIS (KC343637) from D. nobilis complex (Gomes et al. 2013). The ITS sequence obtained in this study showed 99% similarity with that of the strain Ch1 (JF812647), which caused stem canker on cherry in China (Wang et al. 2011). Pathogenicity of the three isolates was performed in the field and under laboratory conditions. Briefly, 30 stems from 5-year-old chestnut trees were wounded with a scalpel on the phloem tissues and inoculated with an inoculum suspension (10⁶ alpha conidia/ml), which were then wrapped with moist cheesecloth and sealed with Parafilm. The wounded phloem tissues of 10 stems were inoculated with sterile distilled water as noninoculated controls. After 2 weeks, necrotic lesions (32 to 55 mm in diameter) developed on inoculated shoots, which resembled the symptoms on infected chestnut shoots in the field. In contrast, control plant tissues did not show any disease symptoms. The pathogen was reisolated from symptomatic shoots and was confirmed as D. nobilis based on morphological and molecular analysis. The experiment was conducted three times. To our knowledge, this is the first report of D. nobilis causing shoot canker and dieback on chestnut in China, and even worldwide. This study provides important information for necessitating new disease-management practices.

Effects of morning vs. evening inoculation on the development of Neonectria ditissima lesions

The study of Neonectria ditissima causing European canker in apple trees is often dependent on controlled inoculation of tree wounds and development of canker lesions. This makes the success of the initial inoculation crucial for time-efficient research. The effect of morning vs. evening inoculation on the successful development of canker lesions was investigated. Ninety-six ‘Royal Gala’ trees were inoculated on six different days (February—March 2017), which covered several different weather conditions. On each inoculation day, 16 trees were inoculated on four rasp and four picking wounds, at 10:00 and 19:00 hours. These were then assessed over the following 8 months for presence of disease symptoms. Irrespective of rain (0—4.5 mm rainfall at the day of inoculation), temperature (average daily temperature of 14.8—21.3˚C), and humidity (average daily relative humidity of 59.8—94.2% rH), neither the day nor the hour of inoculation affected symptom development. However, more inoculated wounds developed symptoms in rasp wounds (91%) than in picking wounds (63%). Under the environmental conditions tested, inoculation timing (date and hour) had little effect on N. ditissima symptom expression.

Neonectria ditissima conidium production and release in planta

Neonectria ditissima, the causal agent of European canker in apples, conidia are available year-round in the Tasman region of New Zealand. Spore production and release in apple trees were quantified during natural and artificial rain events. Rain traps captured spores over 18 rain events (July—August 2017) from European canker lesions (excised and in planta) derived from ‘Royal Gala’ picking wound and rasp wound infections. Rain traps were monitored regularly to determine spore release over time. Differences in the number of conidia produced from lesions on the tree and excised lesions re-mounted in the tree were not significant. Lesions arising from picking wounds produced 7.2 times more spores on average than lesions arising from rasp wounds. Most spores were released within the first hour of rain and total spore release followed a logarithmic curve. Rain duration × volume of water determined the rate of conidia release. Excised lesions are suitable to study the effect of biotic and abiotic drivers on spore production.

Long-term deep-freeze storage of Neonectria ditissima conidium suspensions does not reduce their ability to infect apple trees

Fresh, wild-type conidium suspensions of Neonectria ditissima prepared from sporulating European canker lesions for use in field inoculation trials are not always readily available. The effect of freezing inoculum on the viability of spores and their ability to cause infection in situ was assessed using 15—19 frozen N. ditissima conidium suspensions during 2015—2017. Freshly prepared inoculum and water only were used to inoculate rasp wounds on ‘Braeburn’ (2015), ‘Scilate’/Envyâ„¢ (2016) and ‘Royal Gala’ and ‘Braeburn’ (2017) along with frozen inoculum ranging in age from 1—462 days at concentrations >104 conidia/mL. Lesion development was assessed at approximately monthly intervals. In all experiments, there was no substantial effect of storage time on lesion development although germination in droplets may have decreased during storage. As expected, there was a cultivar effect (2017 data only), with fewer lesions on ‘Braeburn’ compared with ‘Royal Gala’. Long-term deep-freeze storage up to 462 days of N. ditissima conidium suspensions (>104 conidia/mL) did not affect the ability of spores to infect apple trees. Therefore, spore suspensions can be batch-prepared and stored frozen for future experimental use.

3-methylcrotonyl Coenzyme A (CoA) carboxylase complex is involved in the Xanthomonas citri subsp. citri lifestyle during citrus infection.

Citrus canker is a disease caused by the phytopathogen Xanthomonas citri subsp. citri (Xcc), bacterium which is unable to survive out of the host for extended periods of time. Once established inside the plant, the pathogen must compete for resources and evade the defenses of the host cell. However, a number of aspects of Xcc metabolic and nutritional state, during the epiphytic stage and at different phases of infection, are poorly characterized. The 3-methylcrotonyl-CoA carboxylase complex (MCC) is an essential enzyme for the catabolism of the branched-chain amino acid leucine, which prevents the accumulation of toxic intermediaries, facilitates the generation of branched chain fatty acids and/or provides energy to the cell. The MCC complexes belong to a group of acyl-CoA carboxylases (ACCase) enzymes dependent of biotin. In this work, we have identified two ORFs (XAC0263 and XAC0264) encoding for the α and β subunits of an acyl-CoA carboxylase complex from Xanthomonas and demonstrated that this enzyme has MCC activity both in vitro and in vivo. We also found that this MCC complex is conserved in a group of pathogenic gram negative bacteria. The generation and analysis of an Xcc mutant strain deficient in MCC showed less canker lesions in the interaction with the host plant, suggesting that the expression of these proteins is necessary for Xcc fitness during infection.

Pathogenicity of Diaporthe spp. on two blueberry cultivars (Vaccinium corymbosum)

Diaporthe vaccinii causes twig blight and fruit rot and is currently listed as a quarantine organism for the European Union. In the Netherlands, two species from the same genus, Diaporthe eres and Diaporthe rudis, are regularly isolated from blighted twigs of Vaccinium corymbosum. This study compared the pathogenicity of these two species to D. vaccinii. To develop a pathogenicity test a field experiment was performed at an experimental station, testing isolates of D. rudis and D. eres. Most of the isolates tested did not cause statistically significantly larger lesions compared to the control treatment (P > 0.05). In a greenhouse experiment a similar test was conducted, comparing D. vaccinii with the closely related species D. eres. Two cultivars of blueberry, Duke and Liberty, were inoculated using two D. vaccinii and two D. eres isolates. Stem canker lesions caused by D. vaccinii were larger than these caused by D. eres isolates. The majority of the lesions caused by D. vaccinii did not pass the first node of the inoculated shoot, which might be an explanation for the ‘mild’ symptoms of D. vaccinii in V. corymbosum, which appear not to cause crop losses. The results of this study suggest that D. vaccinii may not be a major threat to blueberry production in Europe. Further data is required to be able to conclude whether the quarantine status of D. vaccinii is still appropriate.

Genetic control, combining ability and heritability of resistance to stem canker in cacao (Theobroma cacao L.)

Stem canker caused by Phytophthora palmivora and Phytophthora megakarya is a serious disease of cacao (Theobroma cacao L.). Studies of the genetics of resistance to this disease is important to identify an effective procedure to use in breeding resistant varieties. The objective of this study was to determine mode of inheritance, combining ability and heritability of resistance to stem canker in cacao. A 6 × 6 full diallel mating design was used to generate 36 F1 hybrids which were evaluated for resistance to stem canker with three replications under artificial inoculation with 1 mm2 agar plug taken from actively growing colonies of 10-day old cultures of P. palmivora and P. megakarya. Twelve months later, the canker lesions on outer bark of the stem of the seedlings were measured and the canker lesions inside was also measured after scraping the bark. There were significant (p 50%) of GCA suggest that additive gene effects were more important than non-additive gene effects. The overall direction of results showed that using one of the following parents: P1 (Alpha B36), P4 (T17/524) and P5 (T65/238) as the female produced progenies with better resistance to stem canker disease than when they were used as the male parent. The majority reflected the resistance level of the female parent, suggesting that resistance to stem canker in cacao is influenced by maternal inheritance. Cacao genotypes Alpha B36, T17/524 and T65/238 showed significant negative GCA effects for resistance to stem canker suggesting that these genotypes will transmit the trait to their progenies and therefore recommended for cacao breeding. Narrow sense heritability for stem canker resistance was low indicating large component of dominance effects and influence of environment in inheritance of resistance to stem canker in cacao. However, the very high broad sense heritability (0.99) coupled with large variation within the cacao tree population indicate that progeny testing and within family selection would be appropriate in breeding against stem canker in cacao.

Apple rootstocks may become infected by Neonectria ditissima during propagation

ABSTRACTIntroduction and purpose: The ability of apple rootstocks to become infected by Neonectria ditissima, the cause of European canker, was studied over two years.Materials and methods: Rootstocks B9 and M9 with a size suitable for grafting (6-10 mm stem diameter, termed rootstocks), and smaller sized rootstocks (<5 mm stem diameter, termed transplants) of B9, M9, M26, MM106 and Antonovka were inoculated with N. ditissima at different times, either with contaminated map pins or with spore suspensions. In addition, the rootstocks were either defeathered (side shoots removed), topped (top shoot headed) or both, to create wounds that would normally occur during propagation, while wounds on transplants were made by removing leaves.Results and discussion: One month after inoculation, slightly sunken canker lesions had developed around the inoculation points of the map pins or wounds. No lesions developed on the non-inoculated controls. Map pin inoculation resulted in 30% to 89% infection and spore suspension sprayed on wounds from 5% to 45% infection. When the cankered areas were split open, brown lesions with necrotic tissue due to infection by N. ditissima appeared. The transplants of M9, M26 and MM106 inoculated with contaminated map pins in 2014 developed necrosis on 40% to 67% of the plants, but there were no differences in the incidence or severity among the different types. On the transplants of B9, Antonovka and M9 inoculated in 2015, there was more necrosis on B9 (42%) than on Antonovka (11%) and more sporulating lesions on B9 (29%) than on M9 (9%) or on Antonovka (4%).Conclusion: It can be concluded that rootstocks used for apple trees may become infected by N. ditissima, and wounds should thus be protected during propagation.

Enhanced resistance to citrus canker in transgenic sweet orange expressing the sarcotoxin IA gene

Citrus canker, caused by the bacterial pathogen Xanthomonas citri subp. Citri (Xcc), is a serious disease reported in most citrus-producing areas around the world. Although different levels of field resistance to citrus canker have been reported in sweet oranges, they are usually not sufficient to provide adequate control of the disease. Ectopic over-expression of antibacterial genes is one of the potential strategies to increase plant resistance to bacterial diseases. Previous in vitro results showed that sarcotoxin IA, an antimicrobial peptide isolated from the flesh fly (Sarcophaga peregrina), can be efficient to control different plant pathogenic bacteria, including Xcc. Transgenic “Pera” sweet orange (Citrus sinensis [L.] Osbeck) plants constitutively expressing the sarcotoxin IA peptide fused to the PR1a signal peptide from Nicotiana tabacum for secretion in the intercellular space were obtained by Agrobacterium-mediated transformation using thin sections of mature explants. Citrus canker resistance evaluation in leaves of transgenic and non-transgenic plants was performed through inoculations with Xcc by infiltration and spraying. The Xcc population was up to 2 log unit lower in leaves of the transgenic plants compared to those of non-transgenic controls. Incidence of canker lesions was significantly higher in non-transformed controls (>10 lesions/cm2) than in the transgenic plants (<5 lesions/cm2) after injection infiltration or spraying with Xcc inoculum. Accumulation of sarcotoxin IA peptide in sweet orange tissue did not cause any deleterious effects on the growth and development of the transgenic plants, indicating this approach is suitable to provide resistance to citrus canker.

Phytophthora chlamydospora and P. megasperma associated with root and crown rot of sour cherry in Turkey

Sour cherry is a very important commercial crop of Turkey. In a newly established orchard in Ankara province of Turkey, some of young sour cherry trees did not leaf out or collapsed after a while turning green in spring, 2014. Lateral and hairy roots of symptomatic trees showed poor growth. Occurrence of necrotic and decay tissues girdling the whole roots resulted in tree mortality. Two Phytophthora spp. were isolated from necrotic tissues on taproots and crowns. The pathogens were identified as Phytophthora chlamydospora and P. megasperma based on morphological characteristics and DNA sequences of the internal transcribed spacer region. Pathogenicity of the isolates was tested by stem inoculation to sour cherry seedlings. P. chlamydospora caused canker lesions and killed the seedlings within 4 weeks, while no cankers occurred on stem of the plants inoculated with P. megasperma and on control plants. Also P. megasperma was found as pathogen on roots of sour cherry seedlings in soil infestation test. This is the first report of P. chlamydospora and P. megasperma causing crown and root rot of sour cherry in Turkey. In addition, P. chlamydospora is a new pathogen of sour cherry grafted on Prunus mahaleb.

The Effect of Controlled-release Chlorine Dioxide on the Preservation of Grapefruit

The effect of controlled-release chlorine dioxide (ClO2) gas on the safety and quality of grapefruit was studied. The experiments were run under controlled chamber systems with inoculated fruit, and in boxed fruit under commercial conditions. For the inoculation test, fruit artificially inoculated with either Escherichia coli or Penicillium digitatum, or naturally inoculated Xanthomonas citri ssp. citri (Xcc) (fruits with citrus canker lesions), were incubated in a chamber containing a dose equivalent to 0–60 mg·L−1 of pure ClO2 as an antimicrobial agent. After 24 hours, the microbial population on treated grapefruit was significantly reduced compared with that of control fruit: a dosage of 5 mg·L−1 completely inhibit the growth of E. coli and P. digitatum, but a dosage of 60 mg·L−1 was needed to completely kill Xcc. For the simulated commercial experiment, fruit were harvested in late Oct. 2015 passed through a commercial packing line, and packed in 29 L citrus boxes. ClO2 packets were attached to the top lids with the following five treatments: fast-release, slow-release, slow/fast-release combination (each containing 14.5 mg·L−1 of pure ClO2), double dose fast-release (containing 29 mg·L−1 of ClO2), and control. After 6 weeks of storage at 10 °C (to simulate storage and transportation) + 1 week of storage at 20 °C (to simulate retail marketing), the fruit quality was evaluated. The slow-release treatment at standard dose exhibited the best antimicrobial activity, reducing total aerobic bacterial count and yeast/mold count by 0.95 and 0.94 log colony-forming units (cfu)/g of fruit, respectively, and maintained the best visual, sensory, and overall quality. However, the higher dosage treatments resulted in phytotoxicity as evidenced by peel browning.

First Report of Crown and Root Rot of Plum Caused by Phytophthora megasperma in Turkey

HomePlant DiseaseVol. 101, No. 1First Report of Crown and Root Rot of Plum Caused by Phytophthora megasperma in Turkey PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Crown and Root Rot of Plum Caused by Phytophthora megasperma in Turkeyİ. Kurbetli, G. Sülü, M. Aydoğdu, and İ. Polatİ. KurbetliSearch for more papers by this author, G. SülüSearch for more papers by this author, M. AydoğduSearch for more papers by this author, and İ. PolatSearch for more papers by this authorAffiliationsAuthors and Affiliations İ. Kurbetli G. Sülü M. Aydoğdu İ. Polat , Bati Akdeniz Agricultural Research Institute, 07010 Antalya, Turkey. Published Online:3 Nov 2016https://doi.org/10.1094/PDIS-06-16-0921-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Three- to four-year-old Japanese plum trees (Prunus salicina L.) in a commercial orchard of Antalya Province showed symptoms of decline in July-August 2015. The aerial symptoms were leaf discoloration and twig dieback, and most of the affected trees were leafless. Reddish brown cankers developing from the root to the stem base were observed. Lateral and feeder roots were decayed. Approximately 100 out of 900 plum trees cv. Black Amber and Black Diamond grafted on Myrobalan 29C rootstocks were affected by the disease. Small segments taken from crowns were cut from the margin of lesions and plated onto selective CMA-PARPH medium (Jeffers and Martin 1986), and incubated at 22°C in the dark for 3 to 5 days. Growing colonies were transferred onto carrot agar (CA) (200 ml boiled carrot juice, 800 ml distilled water, 20 g agar) to obtain pure cultures. Isolates were obtained from three of the five symptomatic plum trees that were analyzed. Isolates produced oogonia on CA with a diameter of 31.8 to 46.8 μm (mean 41.4 μm). Plerotic or aplerotic oospores with paragynous antheridia measured 27.6 to 39.6 μm (mean 35.1 μm) in diameter. Nonpapillate and noncaducous sporangia were ovoid, obovoid, obpyriform, ellipsoid or distorted shapes, with rounded and tapered bases. Ovoid and obpyriform sporangia were 45.0 to 90.5 μm long (mean 62.5 μm) and 25.1 to 51.6 μm wide (mean 36.8 μm). Cultures also produced catenulate rounded hyphal swellings. The isolates were identified by morphology as Phytophthora megasperma Drechs. (Erwin and Ribeiro 1996). Morphological identification was confirmed by sequencing of the rDNA region. Genomic DNA was extracted from one isolate, and the internal transcribed spacer (ITS) region was amplified and sequenced using ITS1 and ITS4 primers. Nucleotide sequence of the isolate (KU843555) showed a 99 to 100% similarity with other P. megasperma isolates in GenBank (e.g., HQ643275, EU301166, DQ486668). To test pathogenicity, five plum seedlings cv. Black Amber grafted on Myrobalan 29C rootstocks were transplanted to a 4-liter pot containing autoclaved peat/perlite mixture (3:1, v/v) mixed with wheat grain inoculum at a rate of 4%. Three seedlings grown in sterile mixture without inoculum were used as control. Plants were incubated in a growth chamber for 4 months at 25 ± 1°C. Seedlings were watered twice a week, and in order to keep the seedlings constantly wet, each seedling was placed in a tray filled with 2 to 3 cm of water. At the end of the experiment, canker lesions occurred on taproots and lateral roots, while no cankers developed in the roots of noninoculated plants. The pathogen was reisolated from symptomatic tissues. To our knowledge, this is the first report of P. megasperma causing crown and root rot of plums in Turkey.

Occurrence of a Citrus Canker Strain With Limited Host Specificity in South Texas

In October 2015, a Mexican lime exhibiting citrus canker symptoms was found by the USDA APHIS PPQ in a residential property in Rancho Viejo, Cameron County, Texas. Real-time PCR analysis detected the presence of Xanthomonas citri subsp. citri; USDA APHIS PPQ in Beltsville, MD confirmed this diagnosis. A delimiting survey was initiated and suspect leaf samples were collected and sent to the PPQ Beltsville lab for analysis. By October 2017, leaf samples from 197 trees were confirmed positive for citrus canker, all within a 5-mile radius; in addition, a further 59 symptomatic trees were found and all 256 infected trees (254 Mexican lime, one makrut lime, and one Ponderosa lemon) were removed. Survey data collected on stem lesions suggested the oldest lesions to be between 4 to 6 years old. A host-range study using 12 citrus varieties, including the major commercial varieties grown in Texas, were inoculated with crude leaf extracts from symptomatic leaves by leaf infiltration method. Mexican limes and alemow were the only citrus plants that developed definitive canker lesions. A combination of host range, serological, and molecular tests suggested that this isolate was different from the typical Asiatic strain and more similar to X. citri subsp. citri AW which was only previously reported from Florida on Mexican lime and alemow.

Management of Chinese hickory (Carya cathayensis) trunk canker through effective fungicide application programs and baseline sensitivity of Botryosphaeria dothidea to trifloxystrobin

Trunk canker of Chinese hickory (Carya cathayensis), caused by Botryosphaeria dothidea, is the most important disease threatening production. Although fungicides are applied to control the disease, efficacy is very poor. In this study, fungicides were studied and selected for effective control of trunk canker through assessments of inhibitory activity and application programs. Seventeen fungicides from eight different FRAC groups were tested for their in vitro activity against B. dothidea. Difenoconazole, tebuconazole, and prochloraz (FRAC 3) were the most effective in inhibiting mycelial growth. Trifloxystrobin (FRAC 11) had the most significant inhibitory activity against spore germination with an EC50 of 1.78 ± 0.61 mg/ L. Seven different application programs with three sprays of trifloxystrobin and/or difenoconazole for each program were evaluated at two forest sites over 3 years. Results indicated that the date of the first spray is crucial. If the first spray was applied on March 10, significantly better efficacy was achieved compared to an application on March 20. In 2016, a real-time PCR technique was adopted to help quantify the pathogen and thereby assess canker control. The results suggested a good relationship between gDNA content of B. dothidea in trunk tissues and the development of the canker lesions at both sites. Moreover, a total of 107 single-conidium isolates of B. dothidea were further tested for their sensitivities to trifloxystrobin in conidial germination experiments. In the presence of 100 mg/L SHAM, the EC50 values ranged from 0.29 mg/L to 8.38 mg/L, with an average of 3.16 ± 0.85 mg/L. The range of variation was 28.9-fold. These sensitivity data can be used as a baseline for monitoring putative sensitivity shifts in B. dothidea to trifloxystrobin, a Qo-inhibiting (QoI) fungicide, members of which are commonly considered to be at a high risk for resistance selection.