Detection of Acidovorax valerianellae in corn-salad seeds, seed transmission of the pathogen and disease development in the field

Acidovorax valerianellae , the causal agent of bacterial black spot of corn salad and responsible for severe economic losses to this vegetable in France, was successfully transmitted to corn-salad plants by artificially inoculated seeds in glasshouse and field experiments. In the field experiments, climatic data recorded under plastic tunnels indicated that increasing temperature and relative humidity increased symptom development. To investigate the possible contamination of commercial seedlots of corn salad, a seed test was developed consisting of soaking batches of seeds (five batches each of 5000, 1000, 500 and 100 seeds) overnight at 4°C in distilled sterile water, followed by dilution-plating of seed extracts on TSAV (tryptic soya agar for A. valerianellae ) semiselective medium. Suspected colonies were identified by biochemical and pathogenicity tests or, within 24 h, using antibodies specific to A. valerianellae . Acidovorax valerianellae was detected in three lots. Seed infection levels ranged from 0·10 to 0·89% of contaminated seeds and a single seed carried up to 1800 A. valerianellae colony-forming units.     

A new selective medium for Burkholderia caryophylli, the causal agent of carnation bacterial wilt

A new selective medium (APCA medium) was developed for the isolation of Burkholderia caryophylli , the causal agent of carnation bacterial wilt, from both plants and soil. The optimal concentration and combination of antibiotics was investigated to determine the most selective condition for growing B .  caryophylli . The resultant composition of the medium per litre was: 0·79 g (NH₄)₂SO₄, 1·0 g KH₂PO₄, 0·5 g MgSO₄ · 7H₂O, 0·2 g KCl, 2·0 g D-arabinose, 5 mg crystal violet, 50 mg cycloheximide, 50 mg polymyxin B sulphate, 50 mg ampicillin sodium, 10 mg chloramphenicol, 25 mg blue tetrazolium, and 15 g agar. Plating efficiency ranged from 119 to 174% with an average of 141% compared to that of nutrient agar. The bacterium was successfully isolated from contaminated soil and plant tissues with this medium. Moreover, the medium almost completely inhibited the growth of other plant pathogenic bacteria and soil saprophytes. This selectivity was high enough to detect B . caryophylli in contaminated soil.     

Development of a semiselective medium for isolating Xanthomonas campestris pv. musacearum from insect vectors, infected plant material and soil

A semiselective medium was developed for isolating Xanthomonas campestris pv. musacearum ( Xcm ) from infected banana plants, soil and insect vectors. The new medium was named cellobiose-cephalexin agar (CCA) and it contained (L⁻¹): 1 g yeast extract, 1 g glucose, 1 g peptone, 1 g NH₄Cl, 1 g MgSO₄·7H₂O, 3 g K₂HPO₄, 1 g beef extract, 10 g cellobiose, 14 g agar, 40 mg cephalexin, 10 mg 5-fluorouracil and 120 mg cycloheximide. The medium was evaluated for selectivity using 21 bacterial isolates and for plating efficiency using Xcm . The bacterial isolates included a soilborne Xanthomonas species and three pathogenic Xanthomonas strains that infect cassava, cabbage and beans. Although the plating efficiency of Xcm on CCA was lower (59%) than on non-selective yeast extract peptone glucose agar (YPGA), its selectivity was significantly higher, averaging 60 and 82%, when isolating from banana fruits and soil, respectively. CCA was also superior when isolating Xcm from insect vectors, with selectivity of 48–75%, compared with 8–17% on YPGA. Xanthomonas campestris pv. phaseoli did not grow on CCA, while X. campestris pv. campestris and X. axonopodis pv. manihotis grew, but their colonies were smaller than those of Xcm . Twenty-nine out of 33 suspected Xcm strains isolated from plants, soil and insects using CCA were pathogenic when inoculated onto banana plants, indicating that CCA can be a reliable tool in isolating Xcm populations. The medium should prove useful in studies on ecology, epidemiology and management of the banana bacterial wilt pathogen that is currently ravaging bananas in East and Central Africa.     

Characterization of weeds and rotational crops as alternative hosts of Spongospora subterranea,the causal agent of powdery scab in Israel

Alternative hosts of Spongospora subterranea may allow multiplication and survival of the pathogen over time; thus, host range is important from an epidemiological aspect. Weeds and rotational crops, such as wheat and barley, were sampled from potato fields with a history of powdery scab (PS) and examined for the presence of S. subterranea by root staining followed by microscopic observations and by qPCR analysis after DNA extraction. The pathogen was detected in plants of 16 weed species from eight families and in volunteer plants of potato and wheat. The ability of the pathogen to infect weeds and rotational crops was further examined by artificial inoculations with sporosori in pot experiments. Successful inoculations occurred with 13 weed species from eight families and with 12 rotational crops from five families. The findings of this study indicate a wide host range in Israel; the families Malvaceae and Zygophyllaceae and the following species are reported for the first time as S. subterranea hosts: Solanum elaeagnifolium, Triticum aestivum, Cynodon dactylon, Phalaris paradoxa, Phalaris minor, Setaria verticillata, Rostaria cristata, Sinapis nigra, Arachis hypogaea, Medicago sativa, Astragalus hauraensis, Amaranthus albus, Chenopodium murale, Chenopodium opulifolium, Salsola soda, Malva nicaeensis, Chrysanthemum segetum, Verbesina encelioides, Ammi majus and Tribulus terrestris. Controlling weeds and avoiding the relevant rotational crops observed to be S. subterranea-positive and thus potential hosts, should be taken into consideration in the management of PS, to reduce pathogen inoculum build-up.     

Molecular identification and prevalence of Acidovorax avenae subsp. avenae causing red stripe of sugarcane in China

Red stripe caused by the bacterium Acidovorax avenae subsp. avenae (Aaa) is a disease of sugarcane that is distributed worldwide. In this study, 108 sugarcane leaf samples were collected in 2013–2016 from nine sugarcane‐growing regions in China. Aaa was detected by PCR with specific and novel primers from the 16S–23S rDNA internal transcribed spacer region in 81 of 84 (96%) leaves with red stripe symptoms and in 20 of 24 (83%) leaves without symptoms. Furthermore, Aaa was detected in all nine sampling locations representing six sugarcane‐producing provinces in China. The 101 amplified fragments were cloned and sequenced. The size of the nucleotide sequences varied from 436 to 454 bp and the sequence identity ranged from 89.2% to 100%, suggesting a significant genetic variation among Aaa strains from China. Five major restriction fragment length polymorphism (RFLP) profiles were obtained by in silico and polyacrylamide gel electrophoresis analyses of the PCR products digested with HindIII and EcoRI. The causal agent of sugarcane red stripe was also successfully isolated from a diseased plant and its pathogenicity confirmed by inoculation of healthy sugarcane plantlets and reproduction of disease symptoms. The data showed that Aaa is currently widespread in China, suggesting that control methods should be implemented to limit the impact of red stripe on sugarcane production.     

Comparison of serological, culture, and bait methods for detection of Pythium and Phytophthora zoospores in water

Two immunodiagnostic detection assay procedures were compared with two conventional assays for their sensitivity in detecting propagules of Pythium ultimum var. sporangiiferum , Pythium Group F, Phytophthora cactorum and P. cryptogea in dilution series in sterile distilled water. The most sensitive assay for all four species was the zoospore trapping immunoassay (ZTI). Conventional membrane filtration-dilution plating gave similar results to ZTI with the two Phytophthora spp., but was less sensitive in Pythium detection. Immunodiagnostic dipstick assays and conventional bait tests showed similar sensitivities in the dilution series, and were generally about two orders of magnitude less sensitive than ZTI. The four techniques were also compared for their detection efficacy with water samples collected from horticultural nurseries and in in situ tests of infected root zones of Chamaecyparis , tomato and Chrysanthemum . In these comparisons, ZTI was again the most sensitive test for water samples, although membrane filtration-dilution plating proved to be a more consistent test. Dipstick and baiting assays were the best techniques for in situ testing, and dipsticks provided epidemiologically valuable, quantitative data on pathogen propagule numbers.     

Effect of growth medium, Striga seed burial distance and depth on efficacy of Fusarium isolates to control Striga hermonthica in Burkina Faso

Striga hermonthica is a destructive parasite of cereal crops in the semi‐arid tropical zone. Two greenhouse experiments were conducted at Kamboinsé, Burkina Faso, to investigate the effect of inoculum substrate and location of Striga seeds on the ability of 14 indigenous Fusarium isolates to control the parasite. In Expt 1, Fusarium isolates reduced emerged Striga number, Striga vigour and dry biomass. As a result, sorghum dry biomass and grain yield were enhanced. Inoculum substrate did not influence the ability of Fusarium isolates to control Striga. In Expt 2, Fusarium isolates, substrate and their interaction significantly influenced germination of Striga seeds at both 35 and 50 days after sowing. Isolates grown on compost were more effective at reducing germination of Striga seeds than those grown on chopped sorghum straw. The per cent germination of seeds 50 days after sowing, buried at 5 cm depth, was significantly lower than that of seeds buried at 10 cm. At 10 cm depth, Fusarium isolates still reduced Striga seed germination with respect to the control; horizontal planting distance, 5 or 10 cm from sorghum hills, had no effect.     

The potential for using cyanobacteria (blue-green algae) and algae in the biological control of plant pathogenic bacteria and fungi

Cyanobacteria (blue-green algae) and eukaryote algae occur in freshwater, marine, and terrestrial (soil) habitats. In fact, these microorganisms comprise most of the world's biomass. Although the cyanobacteria are mostly photoautotrophic, some are facultative heterotrophs, capable of growing on certain substrates in darkness. Also, some are non-phototrophic and hence, are obligate heterotrophs. A number of cyanobacteria and eukaryote algae, particularly macroalgae, produce various, biologically active compounds. These include antibiotics which in laboratory tests inhibited bacteria and fungi that incite diseases of humans. In addition, the following fungi which are of interest to plant pathologists, were inhibitedin vitro by substances produced by various cyanobacteria: The saprophytesChaetomium globosum, Cunninghamella blakesleeana, andAspergillus oryzae and the plant pathogensRhizoctonia solani andSclerotinia sclerotiorum. Extracts from seaweeds (macroalgae) sprayed on plants have been reported to reduce the incidence ofBotrytis cinerea (gray mold) on strawberries,Erysiphe polygoni (powdery mildew) on turnips, and damping-off of tomato seedlings. Because many cyanobacteria and algae produce a large number of antibacterial and antifungal materials, are almost never a threat to the environment, and many can be grown in quantity in mass culture, they are suitable candidates for exploitation as biocontrol agents of plant pathogenic bacteria and fungi. Much additional work remains to be done however, to thoroughly evaluate cyanobacteria and algae and their products for this role.     

A standard set of host differentials and unified nomenclature for an international collection of Diplocarpon rosae races

Black spot of rose is distributed throughout the world and is the most serious disease of roses (Rosa spp.) in the outdoor landscape. Resistance breeding has been frustrated by the occurrence of races of the causal pathogen Diplocarpon rosae. Races from Germany, North America and the UK have been characterized and maintained in a pathogenic state. However, these races were characterized using independent sets of host genotypes and are referenced using different nomenclatures. In the present study, a total of 15 D. rosae isolates from these locations, as well as Belgium and Italy, were inoculated to a common set of 15 rose cultivars in replicated, detached leaf trials. Baby Love^TM (cv. Scrivluv) was resistant to all isolates except for one originating from the UK. The rose cultivars Mrs Doreen Pike (Ausdor) and Hansa were resistant to all isolates except for one originating from Minnesota, USA. No rose genotype was universally susceptible. A total of 11 pathogenic races were differentiated based on their unique host ranges and were assigned an international race nomenclature. Nine cultivars are proposed as the first standard set of differential genotypes for characterization of D. rosae races.     

免疫凝聚试纸条和TaqMan探针实时荧光PCR检测西瓜细菌性果斑病菌比较研究

 以西瓜细菌性果斑病菌(Acidovorax avenae subsp.citrulli)菌悬液和田间采集的病组织为试材,研究了免疫凝聚试纸条和实时荧光PCR技术检测的灵敏度和适应性。结果表明,免疫凝聚试纸条检测灵敏度为10⁶ cfu/mL,具有简便、快速、易操作特点,适用于田间快速检测和病害诊断;TaqMan探针实时荧光PCR检测灵敏度达10^3~4 cfu/mL,比传统PCR检测灵敏度(10⁵ cfu/mL)提高了10~100倍,且不需要琼脂糖凝胶电泳、溴化乙锭染色和Southern杂交。但需要昂贵的仪器和试剂,适用于室内检测及相关研究。     

A rapid method for direct detection of Polymyxa DNA in soil

Polymyxa spp. are vectors for a number of economically important soilborne plant viruses. The development of a technique to detect virus and vectors directly in soil would be useful for epidemiological studies and assessment of disease risk prior to planting. A rapid method was developed to extract and quantify Polymyxa spp. DNA from soils. DNA was extracted from three soils infested with Polymyxa betae and three infested with P. graminis using an EDTA lysis buffer in combination with a MagneSil™ DNA extraction kit and Kingfisher™ magnetic particle processor. Primers and probes designed to correspond to sequences within the internal transcribed spacer region 2 (ITS2) of ribosomal DNA enabled recovery and amplification of P. betae and P. graminis DNA using real-time PCR and TaqMan chemistry. For the P. graminis- infested soils, the purity of DNA obtained was sufficient to allow Polymyxa DNA to be amplified without dilution to remove inhibitors, but with P. betae- infested soils, amplification was only achieved if the DNA was diluted 1:10. Using TaqMan PCR, a standard curve was constructed from uninfested soil spiked with known numbers of P. betae cystosori; the quantity of P. betae inoculum from naturally infested soil was then extrapolated from the curve. This technique offers a sensitive method of extracting, detecting and quantifying Polymyxa spp. DNA in soil.     

Comparison of PCR, BIO-PCR, DIA, ELISA and isolation on semiselective medium for detection of Xanthomonas albilineans, the causal agent of leaf scald of sugarcane

Polymerase chain reaction (PCR) and newly designed primers, XAF1/XAR1, were tested for selective detection of the causal agent of leaf scald of sugarcane, Xanthomonas albilineans . The efficiency and reliability of PCR were compared with dot immunobinding assay (DIA), ELISA and classical isolation techniques for detecting X. albilineans in suspensions of pure cells and extracts of field-collected stalk and leaf samples of sugarcane. In addition, classical PCR and BIO-PCR (biological amplification followed by PCR) were compared with isolation on a semiselective agar medium. Classical PCR and BIO-PCR techniques had the advantage of not requiring pathogenicity tests to confirm the identity of colonies tentatively identified as X. albilineans on modified semiselective XAM agar medium. The m-XAM medium and BIO-PCR techniques were the most sensitive; however, the former required seven days whereas the latter required only four days. The BIO-PCR technique was as sensitive as the semiselective medium technique and eliminated the need to conduct any additional tests to confirm the identification.     

Inter- and intraspecific morphometric variation and characterization of Phytophthora isolates from cocoa

Difficulties in the accurate identification of the Phytophthora species responsible for black pod disease of cocoa continue to hamper effective disease control. A re-evaluation of morphological characters ( Brasier & Griffin, 1979 ) and a detailed morphometric analysis of 161 Phytophthora isolates largely associated with black pod disease of cocoa from 17 countries worldwide have shown considerable inter- and intraspecific variation. Stable and more reliable parameters for the identification of the species responsible for the disease have been determined. Colony characteristics such as pattern and growth rate on V8 agar are reasonably characteristic for the cocoa Phytophthora species, and can be used to make preliminary identification to species level. Significant sporangial character variation was found within isolates of species from the same and different sources, highlighting the difficulties in making accurate identification on the basis of raw morphological data. Pedicel length was found to be the most consistent species-linked sporangial characteristic. Cluster plots of length/breadth ratios of sporangia versus reciprocals of sporangial pedicel length clearly separated all isolates into distinct species groups ( P. capsici , P. citrophthora , P. palmivora and P. megakarya ) and can be used reliably to identify accurately those pathogens involved in black pod disease outbreaks.     

Tackling black leaf streak disease and soil fertility constraints to enable the expansion of plantain production to grassland in the humid tropics

In Central Africa, plantain is traditionally grown after a forest fallow. Given increasing urban demand and a lack of forest fallows near urban centres, as well as poor roads and environmental concerns to reduce pressure on forests, research is needed to identify suitable shade, fertility management and cultivars to shift production of plantain to grasslands and to reduce losses to diseases such as black leaf streak disease (BLSD). Effects of light level (full, 67%, 33% light), and nitrogen (N)-amendment on BLSD-tolerant (FHIA-21) and BLSD-susceptible (Batard) cultivars planted on soil from paired grassland and forest sites were determined. BLSD and growth were monitored until 5 months after planting. Three months after planting, leaf area attacked on cultivar FHIA-21 was less than half that on Batard. Plants grown under 33% and 67% light had less leaf area attacked (2.9% and 4.6%, respectively) than those grown in full light (7.3%). Leaf area and dry matter (DM) were higher under shade and when grown on forest soils. Compared to growing BLSD-susceptible plantain on forested land under shade, a shift onto grasslands and a reduction in shade use is predicted to reduce yields. Using cultivar FHIA-21 may limit, but not eliminate, yield loss.     

菜豆普通细菌性疫病菌在土壤和植株残体中的越冬能力

为评估菜豆普通细菌性疫病菌地毯草黄单胞杆菌菜豆致病变种或褐色黄单胞菌褐色亚种在土壤及植物残体中的越冬能力,对采自黑龙江、内蒙古、山西、河北及新疆的18块菜豆生产田的20份土壤及14份植物残体样品进行病原菌分离和鉴定。在MT选择性培养基上有12个土壤样品和13个植株残体样品提取液产生典型的类似黄单胞菌菌落。选取29个分离物进行致病性测定,有27个分离物对菜豆品种"英国红"致病。利用地毯草黄单胞杆菌菜豆致病变种和褐色黄单胞菌褐色亚种的特异性引物X4c/X4e及褐色黄单胞菌褐色亚种特异性引物Xf1/Xf2对29个分离物进行多重PCR检测,其中17个分离物为地毯草黄单胞杆菌菜豆致病变种,10个分离物为褐色黄单胞菌褐色亚种。结果表明,菜豆普通细菌性疫病菌可以在黑龙江、内蒙古、山西、河北的一些菜豆种植区的土壤及植株残体中越冬存活。     

A new selective medium for isolation of Clavibacter michiganensis subsp. michiganensis from tomato plants and seed

A new selective and highly sensitive medium was developed for isolation of Clavibacter michiganensis subsp. michiganensis (Cmm), the causal agent of bacterial canker of tomato, from seed and latently infected plants. The new medium (BCT) proved to be superior to all published semiselective media for Cmm and is denoted as selective medium because of (i) its mean plating efficiency, amounting to ≤89% within 7 days for all 30 Cmm strains from different sources tested; (ii) the high selectivity, because accompanying bacterial species occurring on tomato plants and seed or bacteria obtained from culture collections were inhibited to an extent of 98 to 100%; and (iii) the remarkable detection sensitivity. Thus, 8 CFU of Cmm in field plant homogenates containing 12,750 CFU of accompanying saprophytes were detected on BCT. Under these extreme conditions, all of the published semiselective media (D2, KBT, D2ANX, SCM, mSCM, CMM1, mCNS, and EPPO) gave false-negative results. Either some media were rather toxic and Cmm growth was also inhibited or the other, less toxic media allowed growth of high numbers of saprophytes, so that Cmm growth was suppressed. Exclusively, BCT also supported growth of the closely related C. michiganensis subsp. insidiosus, nebraskensis, and tessellarius. The new medium is recommended for Cmm detection in tomato seed, and in symptomless tomato plantlets, to improve disease control of bacterial canker of tomato.     

泰安市核桃园主要病虫害发生情况及其化学防治用药流程

为明确山东省泰安市核桃园主要病虫害的发生情况及其化学防治的用药流程,于2017—2018年采用田间调查法进行主要病虫害的调查,于2019年采用常规喷雾法对不同防治对象进行化学防治,研究不同用药时间及用药次数下3种农药对核桃的保果效果及对主要病虫害的防治效果,确定用药流程。结果表明,2017年泰安市核桃园主要病虫害以核桃细菌性黑斑病、核桃炭疽病和核桃举肢蛾Atrijuglans aristata为主,造成的病虫果率达83.25%。5月中旬至6月下旬,以核桃细菌性黑斑病和核桃举肢蛾单独发生为主;7月上旬至8月上旬,以核桃细菌性黑斑病和核桃炭疽病单独发生为主;8月中旬至下旬,以核桃炭疽病单独发生和核桃炭疽病+核桃细菌性黑斑病共同发生为主。2018年3种病虫害造成核桃的总体落果率为79.84%,共出现2次落果高峰,即在6月下旬以核桃举肢蛾造成的落果和在8月下旬以核桃炭疽病单独发生和核桃炭疽病+核桃细菌性黑斑病共同发生造成的落果。针对以上3种主要病虫害,在核桃生长期应至少喷药6次,其总体的保果效果和防治效果分别达到96.58%和93.70%,分别显著高于喷药4次的83.72%和70.56%,与喷药8次处理差异不显著。在实际生产中,建议对核桃细菌性黑斑病自5月上旬至8月上旬,每15~20 d用药1次,至少用药6次;对核桃炭疽病自6月下旬至8月上旬,每15~20 d用药1次,至少用药4次;对核桃举肢蛾在5月下旬和7月下旬各用药1次。     

Genomics‐informed design of loop‐mediated isothermal amplification for detection of phytopathogenic Xanthomonas arboricola pv. pruni at the intraspecific level

The objective of this study was to develop a rapid, sensitive detection assay for the quarantine pathogen Xanthomonas arboricola pv. pruni, causal agent of stone fruit bacterial spot, an economically important disease of Prunus spp. Unique targets were identified from X. arboricola pv. pruni genomes using a comparative genomics pipeline of other Xanthomonas species, subspecies and pathovars, and used to identify specific diagnostic markers. Loop‐mediated isothermal amplification (LAMP) was then applied to these markers to provide rapid, sensitive and specific detection. The method developed showed unrivalled specificity with the 79 tested strains and, in contrast to previously established techniques, distinguished between phylogenetically close subspecies such as X. arboricola pv. corylina. The sensitivity of this test is comparable to that of a previously reported TaqMan^? assay at 10³ CFU mL^?1, while the unrivalled speed of LAMP technology enables a positive result to be obtained in <15 min. The developed assay can be used with real‐time fluorescent detectors for quantitative results as well as with DNA‐staining dyes to function as a simplified strategy for on‐site pathogen detection.     

Genetic, phenotypic and pathogenic diversity of Xanthomonas arboricola pv. corylina strains question the representative nature of the type strain

A collection of 31 Xanthomonas arboricola pv. corylina strains isolated from Corylus maxima and C. avellana of different countries were assessed by means of repetitive PCR using ERIC, BOX and REP primer sets and analysis of whole-cell protein extracts; pathogenicity tests to three hazelnut ( C. avellana ) cultivars; and some key biochemical tests. From these studies, the X. arboricola pv. corylina strains were clustered into five and three groups by repetitive PCR and protein analysis, respectively, and by using UPGMA cluster analysis, with two strains forming an outlier group to these. The groups showed a high degree of similarity. Strain membership between the groups designed by the two methods exhibited a high degree of congruence, and diversity between the groups was low. Surprisingly, the two strains originating from C. maxima , that include the type strain NCPPB 935, formed the most distinctive group. No relationship to geographic origin of the strains was evident. All strains proved pathogenic towards three different hazelnut cultivars, although the strains obtained from C. maxima did not incite any significant symptoms on buds and twigs. No other relationships between rep-PCR and whole-cell protein groups and pathogenicity were evident. The distinctiveness of the C. maxima strains was supported further by atypical negative gelatin liquefaction test and reduced quinate metabolism results.