Semi-selective Medium Research Articles

Confirmation of Bacterial Leaf Streak Caused by Xanthomonas oryzae pv. oryzicola on Rice in Madagascar.

Bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola is an important disease of rice. BLS is prevalent in Asia and West Africa, where it was first reported in Nigeria and Senegal in the early 1980s (4). Recently, molecular analysis of strains from Mali (2) and Burkina Faso (5) further confirmed the presence of BLS in West Africa. In Madagascar, BLS symptoms were first reported in the 1980s by Buddenhagen but the causal agent was not unequivocally determined (1). To confirm Buddenhagen's observations using modern molecular typing tools, we surveyed several rice fields in the Antananarivo and Antsirabe districts in March 2013. BLS symptoms were observed on cultivated Oryza sativa grown under both upland and lowland conditions, with a proportion of diseased individuals varying from 30% up to 80%. Symptomatic leaves presenting water-soaked lesions that developed into translucent, yellow streaks with visible exudates at the surface were sampled. One to four centimeter long pieces of diseased leaves were ground using the Qiagen TissueLyser system at 30 rps for 30 s (Qiagen, Courtaboeuf, France). The ground tissue was then macerated in 1 ml of sterile water for 1 h at 4°C. Non-diluted and 10-fold diluted tissue macerates were plated on semi-selective PSA medium (peptone 10 g/liter, sucrose 10 g/liter, glutamic acid 1 g/liter, bacto agar 16 g/liter, actidione 50 mg/liter, cephalexin 40 mg/liter, and kasugamycin 20 mg/liter) and incubated for 3 to 7 days at 28°C. Single, yellow, Xanthomonas-like colonies were isolated on non-selective PSA medium. Diagnostic multiplex PCR was performed on single colonies for pathovar identification (3). Five strains that produced three diagnostic bands corresponding to the X. oryzae pv. oryzicola pattern were further analyzed for pathogenicity on 3-week-old O. sativa cv. Nipponbare plants. Bacteria grown on PSA plates and adjusted to 1 × 108 CFU/ml were infiltrated into rice leaves with a needleless 1-ml syringe (2 × 3 infiltrations per plant and strain). Seven days after incubation in the greenhouse (27 ± 1°C with a 12-h photoperiod), inoculated leaves showed water-soaked lesions that produced yellow exudates corresponding to those initially observed in rice fields and observed for leaves challenged with the X. oryzae pv. oryzicola reference strain BLS256. Symptomatic leaf tissues were ground and plated on non-selective PSA medium, resulting in colonies with typical Xanthomonas morphology that were confirmed as X. oryzae pv. oryzicola by multiplex PCR typing (3), thus fulfilling Koch's postulates. Finally, the five strains were subjected to gyrB sequencing upon PCR amplification using the universal primers XgyrB1F (5'-ACGAGTACAACCCGGACAA-3') and XgyrB1R (5'-CCCATCARGGTGCTGAAGAT-3'). The 743-bp partial gyrB sequences were 100% identical to the gyrB sequence of strain BLS256. As expected, the gyrB sequence of strains KACC10331, MAFF311018, and PXO99A of the X. oryzae pv. oryzae pathovar respectively showed nine, 16, and 10 mismatches in comparison to the Malagasy strains, thus further supporting that they belong to the pathovar oryzicola.

Assessment of spatial distribution of ascospores of Sclerotinia sclerotiorum for regional disease forecasting in carrots

Sclerotinia sclerotiorum causes yield losses in a broad range of plant hosts, including carrot, for which a disease risk forecasting model has been previously developed. Detecting and quantifying airborne ascospores as a component of a sclerotinia rot of carrot (SRC) forecast model is currently accomplished using a Sclerotinia semi-selective medium (SSM) in the blue plate test (BPT) which has a threshold of 5 ascospores per plate. To improve the efficacy of applying the SRC forecast model, data from nine years of air sampling were analysed to determine the number of sampling sites needed to accurately estimate the concentration of airborne ascospores in a region (c. 40 km2) of intense carrot production. In six years, ascospore counts were correlated between sites in 20–100% of the possible pairwise comparisons among air sampling sites. The most consistent relationships among the sites were observed during periods of no detection or detection below the BPT threshold. In most years, periods when ascospore counts surpassed the BPT threshold were consistent among sites, but there were some fields and years when these periods were inconsistent. Based on the observations, one air sampling site should be sufficient to detect ascospores when counts are low, increasing to two or more sites during periods when ascospores are detected near or above threshold levels, and cropping and environmental conditions are conducive to disease.

Evaluación del potencial biofertilizante de bacterias diazótrofas aisladas del cultivo de cacao (Theobroma cacao L.).

The adaptability of cacao (Theobroma cacao L.) to adverse conditions, suggests that the microbiota associated with the rhizosphere of this plant could play an important nutrition and development function. In this paper was valuated the potential of biofertilizer nitrogen fixing bacteria (diazotrophas) isolated by the technique of serial dilutions of cacao soils. Bacteria were isolated on semiselective media (NFb,JMV, LGI, JNFb). The pure isolates were characterized morphologically and biochemically and, additionally the capacity of biological nitrogen fixation (BNF) was evaluated by the Micro-Kjeldahl method. Isolates with better nitrogen fixation ability were selected to evaluate their potential for growth promoter in cocoa plants from seed CCN 51 for 120 days with a control without inoculum. As a result of statistical analysis, the cocoa plants effectively responded to bacterial treatments where growth variables and foliar % N, were significantly increased compared with the control whose response was lower. Highlighting the contribution of isolated DKA2J24 and DKB3J73 presumably genus Burkholderia sp., and DKA1L3 like genus Gluconacetobacter sp. all with great potential as biofertilizers in the bioassay test.

First Report of Burkholderia glumae Causing Bacterial Panicle Blight on Rice in Ecuador.

Rice (Oryza sativa L.) is one of the leading crops and the basis of most diets in Ecuador and other countries. Diseases such as bacterial panicle blight (BPB), also known as seedling rot or grain rot, have the potential to threaten rice production worldwide. Burkholderia glumae, a causal agent of BPB, has severely affected the rice industry in many countries of Africa, Asia, and the Americas (1,2,4), but no report of this bacteria in Ecuador can be found in the literature. Rice plantations showing BPB-like symptoms including upright panicles with stained and vain grains were spotted in Palestina city, one of Ecuador's most extensive rice areas, in July 2013, but similar symptoms have been observed in the region since early 2012. Six symptomatic plants from two different groves were collected. Samples were plated on the semi-selective medium S-PG (KH2PO4 1.3 g, Na2HPO4 1.2 g, (NH4)2SO4 5 g, MgSO4·7H2O 0.25 g, Na2MoO4·2H2O 24 mg, EDTA-Fe 10 mg, L-cystine 10 μg, D-sorbitol 10 g, pheneticillin potassium 50 mg, ampicillin sodium 10 mg, cetrimide 10 mg, methyl violet 1 mg, phenol red 20 mg, agar 15 g/liter distilled water) and axenic colonies were transferred to potato dextrose agar (PDA) to test for fluorescence (3). Colonies of the potential pathogen were 1 mm, circular, entire margin, with a smooth and shiny surface. When cultured in PDA, isolates showed a moist texture, dull yellow color, and displayed fluorescence with exposure to UV light. Cells were bacterial gram-negative rods of 1 to 2 × 0.5 μm. Twelve presumptive isolates were submitted to biochemical tests (API 20NE). The biochemical profile (APIWEB) showed that all the isolates belonged to the Burkholderia genus with a 99.9% similarity. To determine the bacterial species, colonies were submitted to ELISA tests using specific antibodies for B. glumae from Agdia, Inc. The two isolates that were positive for B. glumae were sequenced using a part of the 16s rDNA amplified by the primers 536F: 5'-GTGCCAGCMGCCGCGGTAATAC-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'. The obtained sequences (deposited into GenBank as KF601202) shared 100% similarity with several B. glumae strains after a BLAST query. Isolates were then diluted to 108 UFC/ml and used to inoculate healthy rice plants. Inoculated plants produced BPB-like symptoms including upright panicles with stained vain grains and the bacterium was re-isolated from symptomatic plants. To the best of our knowledge, this is the first report of B. glumae in Ecuador. Further research is ongoing to identify and determine the pathogenicity of the remaining Burkholderia strains that tested negative for B. glumae.

Interactions of seedborne bacterial pathogens with host and non-host plants in relation to seed infestation and seedling transmission.

The ability of seed-borne bacterial pathogens (Acidovorax citrulli, Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato, Xanthomonas euvesicatoria, and Pseudomonas syringae pv. glycinea) to infest seeds of host and non-host plants (watermelon, tomato, pepper, and soybean) and subsequent pathogen transmission to seedlings was investigated. A non-pathogenic, pigmented strain of Serratia marcescens was also included to assess a null-interacting situation with the same plant species. Flowers of host and non-host plants were inoculated with 1×106 colony forming units (CFUs)/flower for each bacterial species and allowed to develop into fruits or umbels (in case of onion). Seeds harvested from each host/non-host bacterial species combination were assayed for respective bacteria by plating on semi-selective media. Additionally, seedlots for each host/non-host bacterial species combination were also assayed for pathogen transmission by seedling grow-out (SGO) assays under greenhouse conditions. The mean percentage of seedlots infested with compatible and incompatible pathogens was 31.7 and 30.9% (by plating), respectively and they were not significantly different (P = 0.67). The percentage of seedlots infested with null-interacting bacterial species was 16.8% (by plating) and it was significantly lower than the infested lots generated with compatible and incompatible bacterial pathogens (P = 0.03). None of the seedlots with incompatible/null-interacting bacteria developed symptoms on seedlings; however, when seedlings were assayed for epiphytic bacterial presence, 19.5 and 9.4% of the lots were positive, respectively. These results indicate that the seeds of non-host plants can become infested with incompatible and null-interacting bacterial species through flower colonization and they can be transmitted via epiphytic colonization of seedlings. In addition, it was also observed that flowers and seeds of non-host plants can be colonized by compatible/incompatible/null-interacting bacteria to higher populations; however, the level of colonization differed significantly depending on the type of bacterial species used.

Semi-selective culture medium for Exserohilum turcicum isolation from corn seeds

Northern corn leaf blight, caused by Exserohilum turcicum (Et), is a disease of widespread occurrence in regions where corn, sweetcorn and popcorn are grown. This disease has great potential to cause damage and has been studied for years, but the association of its causal agent with seeds remains unconfirmed. Thus, the availability of a sensitive method to detect and quantify the inoculum in seeds, even at low incidence, is essential. The aim of this study was to develop a method to detect and quantify the presence of the fungus infecting and infesting corn and popcorn seeds. Artificially and naturally infected seeds were employed to develop the medium. The semi-selective medium was composed of carbendazim (active ingredient) (60 mg/L), captan (30 mg/L), streptomycin sulfate (500 mg/L) and neomycin sulfate (600 mg/L) aggregated to the medium lactose casein hydrolysate agar medium. By using this, Et was detected in naturally infected corn seeds, showing 0.124% incidence, in four out of ten analyzed samples. In addition, 1.04 conidia were detected per infested seed. By means of isolation, pathogenicity test, morphological characterization and comparison with descriptions of the species in the literature, the fungus isolated from the seeds was confirmed to be Et. Both infection and infestation were considered low; thus, for studies of Et detection in corn seeds, the use of semi-selective medium and more than 1,200 seeds/sample is suggested.

Identificação e quantificação de fungos associados a sementes de azevém (Lolium multiflorum Lam.)

O trabalho teve como objetivos, identificar e quantificar os fungos associados a sementes de azevém, comparar a incidência em diferentes meios de cultura, e determinar o número de escleródios de Claviceps purpurea presentes em amostras de sementes. Foram analisadas 37 amostras de sementes de azevém provenientes de municípios do Rio Grande do Sul, Santa Catarina e Paraná. As sementes foram plaqueadas em três meios de cultura: BDA, semi-seletivo de Reis e semi-seletivo de Segalin & Reis, analisando-se a incidência dos fungos. Para detecção de C. purpurea, foram pesados 100g de sementes por amostra e, através de exame visual, foi determinado o número de escleródios. Os fungos detectados foram Alternaria alternata, Bipolaris sorokiniana, Drechslera spp., D. siccans, Fusarium graminearum, Fusarium spp., Aspergillus spp. e Penicillium sp. A incidência de A. alternata variou de 0,0% a 33,7% e freqüência de 89,2% nas amostras analisadas. Para B. sorokiniana a incidência foi de 0,0% a 2,2% e frequência de 62,2%, Drechslera spp., apresentou incidência de 0,0% a 40,3% e frequência de 78,4%. D. siccans a incidência foi de 0,1% a 20,0% e frequência de 100%.Para Fusarium spp., e F. graminearum a incidência foi de 0,0% a 31,0% e 0,0% a 11,3% e frequência de 81,1% e 64,9%, de 0,0% a 43,7% de incidência e 94,6% de frequência para Aspergillus spp. e Penicillium sp. com incidência entre 0,0% a 51,7% e frequência de 91,9%, respectivamente. O fungo C. purpurea foi encontrado em 81,1% das amostras em estudo.

The Italian inter-laboratory study on the detection of Pseudomonas syringae pv. actinide

A severe form of bacterial canker of kiwifruit, caused by Pseudomonas syringae pv. actinidiae (Psa), has been detected in all the main areas of cultivation of kiwifruit (Actinidia deliciosa and A. chinensis). Since 2010 several research groups have been assessing methods and procedures to detect and identify Psa, both from symptomatic and symptomless host material. In 2011, a study to compare Psa diagnostic methods was performed with reference to Psa strains and related pathovars, and with plant extracts or DNA obtained from healthy and naturally infected leaves, pollen or wood. The study revealed the strengths and the weaknesses of the assessed methods. The procedure included screening tests for Psa detection and for identification of Psa colonies. The methods assessed were bacterial isolation on generic and semi-selective media, PCR analysis (single, duplex and rep-PCR assay, the latter for identification only). The results highlighted the best performance of semi-selective with respect the generic media; the usefulness of the direct-PCR as screening tests for Psa detection; and the greater specificity of duplex-PCR and sensitivity of simple-PCR. The use of semi-selective medium for isolation and of two PCR-based methods - in parallel - for Psa detection are suggested. Both rep-PCR and duplex-PCR, were found to be specific, and are recommended as an identification test for this pathogen.

Development of a semi-selective medium for improved isolation of the turfgrass dollar spot pathogen Sclerotinia homoeocarpa from host tissues

Dollar spot, caused by Sclerotinia homoeocarpa, is one of the most economically devastating diseases of amenity turfgrasses worldwide. The pathogen is readily isolated from active lesions, but detection from seed, dormant host tissue and other plant debris that may serve as a source of primary inoculum is difficult. A semi-selective medium was developed to enhance isolation of S. homoeocarpa. Various fungicides used on turfgrass, the pH indicator dye bromophenol blue, and two pH levels, were assessed for their effects on, and in the case of bromophenol blue, response to growth of S. homoeocarpa and contaminant fungi frequently isolated from field and seed samples. Amendment of the medium to pH 4 promoted growth of S. homoeocarpa in the absence of fungicides and enhanced pathogen growth relative to contaminant isolates on medium amended with 0.1 μg mL−1 triticonazole or 5 μg mL−1 azoxystrobin. The growth rate of S. homoeocarpa on these three media, as determined by in vitro radial growth assays, was consistent for many isolates representing the majority of S. homoeocarpa vegetative compatibility groups. Isolation efficiency on the pH-amended media was tested against antibiotic-amended potato dextrose agar from field samples and artificially inoculated turfgrass seed. In both cases, the medium amended to pH 4 and containing 5 μg mL−1 azoxystrobin significantly decreased contaminant growth with no adverse effects on recovery of S. homoeocarpa in comparison with antibiotic-amended PDA. The enhanced specificity of this medium will be a useful tool for selective detection of S. homoeocarpa and identification of sources of initial inoculum for dollar spot development.

Sensitive quantitative detection of Ralstonia solanacearum in soil by the most probable number-polymerase chain reaction (MPN-PCR) method

We developed a sensitive quantitative assay for detecting Ralstonia solanacearum in soil by most probable number (MPN) analysis based on bio-PCR results. For development of the detection method, we optimized an elution buffer containing 5g/L skim milk for extracting bacteria from soil and reducing contamination of polymerase inhibitors in soil extracts. Because R. solanacearum can grow in water without any added nutrients, we used a cultivation buffer in the culture step of the bio-PCR that contained only the buffer and antibiotics to suppress the growth of other soil microorganisms. To quantify the bacterial population in soil, the elution buffer was added to 10g soil on a dry weight basis so that the combined weight of buffer, soil, and soil-water was 50g; 5mL of soil extract was assumed to originate from 1g of soil. The soil extract was divided into triplicate aliquots each of 5mL and 500, 50, and 5μL. Each aliquot was diluted with the cultivation buffer and incubated at 35 °C for about 24h. After incubation, 5μL of culture was directly used for nested PCR. The number of aliquots showing positive results was collectively checked against the MPN table. The method could quantify bacterial populations in soil down to 3cfu/10g dried soil and was successfully applied to several types of soil. We applied the method for the quantitative detection of R. solanacearum in horticultural soils, which could quantitatively detect small populations (9.3cfu/g), but the semiselective media were not able to detect the bacteria.

Long‐term survival of Acidovorax citrulli in citron melon (Citrullus lanatus var. citroides) seeds

Long‐term survival of Acidovorax citrulli in citron melon (Citrullus lanatus var. citroides) seeds was investigated. Citron melon seed lots infected with A. citrulli were generated in the field by inoculating either the pistils (stigma) or pericarps (ovary wall) of the female blossoms. Seventeen A. citrulli isolates from 14 different haplotypes belonging to two different groups (group I and II) were used for inoculation. After confirming that 100% of seed lots were infected, they were stored at 4°C and 50% RH for 7 years. After storage, the viability of A. citrulli cells from individual lots was determined by plating macerated seeds on semiselective medium as well as growing seeds for 14 days and scoring for bacterial fruit blotch symptoms. The type of A. citrulli isolate (group I or group II) used did not significantly influence bacterial survival. However, A. citrulli survival was significantly greater in seed lots generated via pistil inoculation (52·9 and 29·4%) than via pericarp inoculation (23·5 and 17·6%). Repetitive extragenic palindrome (rep)‐PCR on A. citrulli isolated from citron melon seed lots after storage displayed similar fingerprinting patterns to those of the reference strains originally used for blossom inoculation, indicating that cross‐contamination did not occur. The results indicate that A. citrulli may survive/overwinter in citron melon seeds for at least 7 years and bacterial survival in seed was influenced more by method of blossom inoculation than by the type of bacterial isolate.

Evaluation of Air Sampling and Detection Methods to Quantify Airborne Ascospores of Sclerotinia sclerotiorum.

Detection and quantification of airborne ascospores as a component of the Sclerotinia rot of carrot (SRC) forecast model is currently accomplished using the blue plate test (BPT), which uses Sclerotinia semiselective medium (SSM). A quantitative polymerase chain reaction (qPCR) assay was developed to reduce the time to specifically quantify ascospores of Sclerotinia sclerotiorum from air samples collected using a Burkard Multi-Vial Cyclone Sampler. The qPCR assay was highly sensitive and detected DNA from 0.5 to 5 × 104 ascospores within a linear range (R2 = 0.99). The qPCR assay was used to quantify ascospores of S. sclerotiorum in air samples collected over three growing seasons. Initial SRC disease was observed 8 and 34 days following detection of 9.5 and 2 ascospores m-3 of air, respectively. Results from air samples collected using an Andersen N6 Sampler and the qPCR assay were compared with the BPT. Ascospore counts from a Burkard Sampler coupled with the qPCR assay and the BPT followed similar trends. In general, fewer ascospores were detected and bioaerosol sampling efficiency was low using an Anderson Sampler. Three days were required to confirm the number of ascospores using SSM in the BPT and with an Andersen Sampler, whereas results from a Burkard Sampler coupled with the qPCR assay can provide results within 5 h of air sampling. The choice of method will depend on the available resources.

Quantitative Molecular Detection of Xanthomonas hortorum pv. carotae in Carrot Seed Before and After Hot-Water Treatment.

Molecular assays to detect and quantify DNA from viable cells of the seedborne pathogen Xanthomonas hortorum pv. carotae in carrot seed were developed and evaluated for use on nontreated and hot-water-treated seed lots. Both a TaqMan real-time polymerase chain reaction (PCR) assay and a loop-mediated isothermal amplification (LAMP) dilution endpoint assay detected and quantified DNA from viable pathogen cells after treatment of carrot seed washes with the live-dead discriminating dye propidium monoazide (PMA). The detection limits of the assays were approximately 101 CFU for pure cultures of X. hortorum pv. carotae, and 102 to 103 CFU/g seed from naturally infested carrot seed lots. X. hortorum pv. carotae in and on carrot seed was killed by soaking the seed in hot water (52°C for 25 min), and a subsequent PMA treatment of these hot-water-treated seed washes suppressed detection of the pathogen with both the real-time PCR and LAMP assays. For 36 commercial seed lots treated with PMA but not hot water, regression of colony counts of X. hortorum pv. carotae measured by dilution plating on a semiselective agar medium versus estimates of pathogen CFU determined by the molecular assays resulted in significant (P ≤ 0.05) linear relationships (R2 = 0.68 for the real-time PCR assay and 0.79 for the LAMP assay). The molecular assays provided quantitative estimates of X. hortorum pv. carotae infestations in carrot seed lots in <24 h, which is a significant improvement over the 7 to 14 days required to obtain results from the traditional dilution-plating assay.

First Report of Xanthomonas citri pv. citri Causing Asiatic Citrus Canker in Burkina Faso.

Citrus canker, caused by Xanthomonas citri pv. citri, is a bacterial disease of economic importance in tropical and sub-tropical citrus-producing areas (EPPO-PQR online database). X. citri pv. citri causes severe infection in a wide range of citrus species, and induces erumpent, callus-like lesions with water-soaked margins leading to premature fruit drop and twig dieback. It has consequently been subjected to eradication efforts and international regulations. It was first described on the African continent in South Africa at the beginning of the 20th century, from which it was eventually eradicated. Since 2006, several outbreaks caused by phylogenetically diverse strains of X. citri pv. citri have been reported from several African countries (Ethiopia, Mali, Senegal, and Somalia). In July 2011, citrus canker in Burkina Faso was suspected in the area adjacent to the Sikassso Province of Mali where X. citri pv. citri has been confirmed. In November and December 2012, leaves of clementine (Citrus clementina), lemon (C. limon), Volkamer lemon (C. volkameriana), sweet orange (C. sinensis), tangelo (C. paradisi× C. reticulata), and mandarin (C. reticulata) were collected from orchards with trees showing symptoms of citrus canker in the Comoé, Houet, and Kénédougou provinces of Burkina Faso. Isolations performed using KC semi-selective medium (4) recovered 45 Xanthomonas-like strains. All Xanthomonas-like strains were tentatively identified as X. citri pv. citri by PCR (4/7 primers) using IAPAR 306 and sterile distilled water as the positive and negative controls, respectively (3). Among these, two strains (LK4-4 and LK4-5) produced a 'fuscans'-like brown diffusible pigment, a phenotype never reported previously for X. citri pv. citri. MultiLocus Sequence Analysis targeting six housekeeping genes (atpD, dnaK, efp, gltA, gyrB, and lepA) (1,2) fully identified seven strains from Burkina Faso (LJ301-1, LJ303-1, LK1-1, LK2-6, LK4-3, LK4-4, and LK4-5) as X. citri pv. citri (and not to any other Xanthomonas pathovars pathogenic to citrus or host range-restricted pathotypes of pathovar citri), and more specifically as sequence type ST2 which is composed mostly of pathotype A strains of X. citri pv. citri (2). The same seven strains were inoculated to at least four leaves of each of grapefruit cv. Henderson, Mexican lime SRA 140 (C. aurantifolia), Tahiti lime SRA 58 (C. latifolia), and sweet orange cv. Washington Navel, using a detached leaf assay (2). All strains developed typical erumpent, callus-like tissue at wound sites on all citrus species inoculated. No lesions developed on the negative control (sterile 10 mM tris buffer). Koch's postulate was fulfilled after reisolation of Xanthomonas-like yellow colonies from symptoms on Mexican lime produced by the seven strains. Boiled bacterial suspensions were assayed by PCR with 4/7 primers (3) and produced the expected 468-bp amplicon in contrast with the PCR negative control. To our knowledge, this is the first report of X. citri pv. citri in Burkina Faso. Citrus canker-free nurseries and grove sanitation should be implemented for reducing the prevalence of Asiatic canker in Burkina Faso and a thorough survey of citrus nurseries and groves in the region should be conducted.

First report of Curtobacterium flaccumfaciens pv. flaccumfaciens on soybean in Brazil

Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) causes bacterial wilt on beans (Phaseolus vulgaris) and bacterial tan spot on soybeans (Glycine max). Cff was detected on beans in Brazil in 1995. Plants of commercial and experimental fields of soybean with typical symptoms of the disease were collected in the State of Parana, Brazil, during the 2011/2012 growing season. The causal agent was identified as Cff by isolation from symptomatic leaves on CNS semi-selective medium, artificial inoculation test and re-isolation in soybean and bean, Gram staining test, solubility in KOH, and by PCR. This is the first report of Cff on soybean in Brazil.

Risk of dissemination of Clavibacter michiganensis ssp. sepedonicus with potato waste

To estimate the risk of dissemination of Clavibacter michiganensis ssp. sepedonicus through potato residues from processing industries, the various processes and the usage of residues from plants from different processing branches were analysed with regard to the effect they can have on the pathogen. The residues were classified into different risk categories, from category 0 (no risk of dissemination) to category 4 (high risk of dissemination). Residues not heated during processing and used in agriculture, e.g., as fertilizer, were pooled in the highest risk category 4. Residues that were sanitised before use in agriculture, e.g., by composting or pasteurisation, were still classified as probably high risk (risk category 3), as no information on these treatments concerning the inactivation of the pathogen was available so far. Therefore the effect of composting and pasteurisation under varying conditions was tested on samples (ready-made compost mould) contaminated with Clavibacter michiganensis ssp. sepedonicus. Viable bacteria could be extracted after all experiments via bioassay on eggplants, and cultivated on semi-selective media from plant sap forming characteristic colonies. The viable pathogen could be extracted after composting for 6 days at maximum temperatures at 70 °C, 13 days at 55 °C and 90 min pasteurisation at 70 °C. It can be concluded that these sanitation treatments are not sufficient to inactivate Clavibacter michiganensis ssp. sepedonicus and the previous classification of treated residues in category 3 (probably high risk) could thus be confirmed.

Development of a Semiselective Medium for Detection of Nalanthamala psidii, Causal Agent of Wilt of Guava.

Guava wilt, caused by Nalanthamala psidii, has become an important disease of guava (Psidium guajava) in Taiwan since the 1970s. This study was conducted to develop a semiselective medium for detecting N. psidii in soil and in tissues of diseased guava trees. Among 9 carbon and 21 nitrogen compounds tested in a modified Czapek-Dox medium, the most effective carbon and nitrogen sources for mycelial growth of N. psidii were sucrose and glycine, respectively. Among eight fungicides tested, iprodione at 5 μg ml-1 and azoxystrobin at 1 μg ml-1 were the most effective fungicides for detection of N. psidii in artificially infested soil or in naturally infected guava debris. Based on the requirement for carbon and nitrogen sources and response to fungicides, a semiselective medium designated as modified sucrose-glycine semiselective medium (mSGSSM) was developed for isolation of N. psidii, using the modified Czapek-Dox medium containing 3% sucrose, 0.3% glycine, iprodione at 5 μg ml-1, azoxystrobin at 1 μg ml-1, streptomycin at 200 μg ml-1, and neomycin at 200 μg ml-1. Colonies of N. psidii on mSGSSM at 30°C for 5 to 10 days were white to orange with sparse aerial hyphae. N. psidii was detected more accurately and efficiently on mSGSSM than on other media, including potato dextrose agar, modified Nash-Snyder medium, and modified Czapek-Dox medium. This semiselective medium is effective in detection of N. psidii from various parts of diseased guava trees and in soil; therefore, it would be a useful medium for etiological, ecological, and epidemiological studies of guava wilt.

First Report of Verticillium Wilt Caused by Verticillium nonalfalfae on Tree-of-Heaven (Ailanthus altissima) in Ohio.

Verticillium wilt of the highly invasive tree-of-heaven [Ailanthus altissima (Mill.) Swingle], caused by Verticillium nonalfalfae Interbitzin et al. (1), formerly classified as V. albo-atrum Reinke and Berthold, has been reported in the United States from two states: Pennsylvania (2) and Virginia (3). Infected A. altissima in both states exhibited similar symptoms of wilt, premature defoliation, terminal dieback, yellow vascular discoloration, and mortality. In June 2012, the second author observed dead and dying A. altissima trees in southern Ohio (Pike County) that exhibited symptoms similar to those on diseased A. altissima trees in Pennsylvania and Virginia. Samples were collected from stems of three symptomatic A. altissima trees and sent to Penn State for morphological and molecular identification. Immediately upon arrival, samples were surface-disinfected and plated onto plum extract agar (PEA), a semi-selective medium for Verticillium spp., amended with neomycin and streptomycin (2). The samples yielded six isolates, two from each of the three symptomatic trees, all of which were putatively identified as V. nonalfalfae based on the presence of verticillate conidiophores and formation of melanized hyphae. DNA was extracted from three isolates and molecular analyses performed using known primers (1) coding for elongation factor 1-alpha (EF), glyceraldehyde-3-phosphate dehydrogenase (GPD), and tryptophan synthase (TS). A BLAST search generated sequences that revealed 100% similarity to V. nonalfalfae for all three protein coding genes among the three Ohio isolates and reference sequences from Ailanthus, including isolates VnAaPA140 (GenBank Accession Nos. KC307764, KC307766, and KC307768) and VnAaVA2 (KC307758, KC307759, and KC307760), as well as isolate PD592 from potato (JN188227, JN188163, and JN188035), thereby confirming taxonomic placement of the Ohio Ailanthus isolates among those recovered from Ailanthus in Pennsylvania and Virginia. Aligned sequences from one representative isolate, VnAaOH1, were deposited into GenBank as accessions KC307761 (EF), KC307762 (GPD), and KC307763 (TS). In August 2012, the pathogenicity of all six isolates was confirmed by root-dipping 10 healthy 3-week-old A. altissima seedlings (seeds collected in University Park, PA) into conidial suspensions of 1 × 107 cfu/ml, wherein all inoculated seedlings wilted and died within 4 and 9 weeks, respectively. V. nonalfalfae was reisolated from all inoculated seedlings; control seedlings inoculated with distilled water remained asymptomatic. Ohio is the third state from which V. nonalfalfae has been reported to be pathogenic on A. altissima. If V. nonalfalfae proves to be widespread, it may represent a natural biocontrol for the invasive A. altissima. Also, since USDA APHIS evaluates and regulates new potential biocontrol agents on a state-by-state basis, it is important to document each state in which V. nonalfalfae is killing A. altissima, so that in-state inoculum can be used for biocontrol efforts, simplifying the regulatory process.

First Report of Xanthomonas citri pv. citri-A Causing Asiatic Citrus Canker in Mayotte.

Asiatic citrus canker, caused by Xanthomonas citri pv. citri, is a bacterial disease of major economic importance in tropical and subtropical citrus-producing areas. X. citri pv. citri pathotype A can cause severe infection in a wide range of citrus species and induces erumpent, callus-like lesions with water-soaked margins evolving to corky cankers and leading to premature fruit and leaf drop and twig dieback on susceptible/very susceptible cultivars. A chlorotic halo is typically visible around canker lesions on leaves and young fruit, but not on mature fruit and twigs. This quarantine organism can strongly impact both national and international citrus markets. Long distance dispersal is mainly through infected propagative material. Asiatic citrus canker occurs on most islands in the Southwest Indian Ocean region (Comoros, Mauritius, Reunion, Rodrigues, and Seychelles islands), but was not yet reported in Mayotte (EPPO-PQR available at http://www.eppo.int ). In May 2012, typical canker-like symptoms were observed on sweet orange (Citrus sinensis) groves on Mtsamboro islet and soon after on the main island of Mayotte, mostly on sweet oranges, but also on Tahiti limes (C. latifolia) and mandarins (C. reticulata). Eighty-one Xanthomonas-like strains were isolated using KC semi-selective medium (4) from disease samples collected from both commercial groves and nurseries on different Citrus species located all over the island. Sixteen Xanthomonas-like isolates were tentatively identified as X. citri pv. citri based on a specific PCR assay with 4/7 primers (3). All strains but the negative control, sterile water, produced an amplicon of the expected size similar to X. citri pv. citri strain IAPAR 306 used as positive control. Multilocus sequence analysis targeting six housekeeping genes (atpD, dnaK, efp, gltA, gyrB, and lepA) (1,2) fully identified three strains from Mayotte (LJ225-3, LJ228-1, and LJ229-11) as X. citri pv. citri (and not other xanthomonad pathovars pathogenic to citrus or host range-restricted pathotypes of pathovar citri), and more specifically as sequence type ST2 composed of pathotype A strains of X. citri pv. citri (2) (including all strains from the Southwest Indian Ocean region). Eight strains were inoculated by a detached leaf assay (2) to Mexican lime SRA 140 (C. aurantifolia), Tahiti lime SRA 58, sweet orange cv. Washington Navel, alemow SRA 779 (C. macrophylla), and tangor cv. Ortanique (C. reticulata × C. sinensis) and developed typical erumpent, callus-like tissue at wound sites for all Citrus species, fulfilling Koch's postulates. Xanthomonas-like yellow colonies were reisolated from symptoms produced by the eight strains inoculated on Mexican lime. Boiled bacterial suspensions were assayed by PCR with 4/7 primers (3) and produced the expected 468-bp amplicon in contrast with the negative control (sterile water). No lesions developed on the negative control consisting of inoculations by 10 mM tris buffer (pH 7.2). Citrus canker-free nurseries and grove sanitation should be implemented for decreasing the prevalence of Asiatic canker in this island territory.

English

The aim of this work was to develop a semi-selective medium for recovery and enumeration of Pichia guilliermondii strain Z1, a reliable biocontrol agent against postharvest pathogens of citrus fruit, and to assess the population dynamic of this antagonistic yeast on orange fruit in relation to incubation temperature and time of incubation. PDA is the basal medium used in this study which allows the antagonistic strain Z1 source of carbon and nutrients. Different chemicals (thiophanate-methyl, thiabendazole, thiram and imazalil) and antibiotics (hygromycin, tetracyclin, ampicillin and chloramphenicol) have been tested individually and based on the plating efficiency up to 90%, 40 combinations have been assayed between antibiotics and fungicides. Ten combinations proved to be highly selective against citrus pathogens and laboratory microflora, but only one consisting of tetracyclin (1 g/L) and thiabendazole (60 mg/L) was retained based on plating efficiency up to 99%, total selectivity against laboratory microflora, epiphytic microflora from washed orange fruit and lower cost. The semi-selective medium TET-TBZ-PDA has been used to assess the impact of temperature and incubation time on the survival of strain Z1. It appears that population density was significantly influenced by both factors and the highest population size was recorded at 25°C followed by 5 and 35°C, respectively. This strain required a time of adaptation before entering the exponential growth phase with a maximum growth observed at 25°C relative to others. The semi-selective medium TET-TBZ-PDA could be an efficient and valuable way to track the population density of this strain on the surface of orange when applied pre-or postharvest. This semi-selective medium may also aid in reaching a population density allowing a better efficiency in relation to environmental conditions. Key words: Antibiotics, biocontrol, citrus, fungicides, quantification, plating efficiency,Pichia guilliermondii strain Z1, semi-selective medium.