Semiselective Agar Medium Research Articles

Assessment of Two Novel Host-Derived Beauveria bassiana (Hypocreales: Cordycipitaceae) Isolates Against the Citrus Pest, Diaphorina citri (Hemiptera: Liviidae).

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), vectors 'Candidatus Liberibacter spp.', the causative agent of Citrus Greening Disease (CGD) or Huanglongbing (HLB). Managing populations of psyllids in the Lower Rio Grande Valley (LRGV), TX, United States is imperative given a continuous increase in HLB-positive trees. A component of integrated pest management (IPM) program is the use of strains of entomopathogenic fungi for the biological control of D. citri. In an attempt to find endemic strains of entomopathogenic fungi that grow favorably under LRGV environmental conditions and naturally infect D. citri, psyllids were collected from local residential areas, surface sterilized, and plated on a semi-selective agar medium. Collection of over 9,300 samples from 278 sites throughout the LRGV led to the positive identification of two Beauveria bassiana (Balsamo-Crivellii) Vuillemin (Hypocreales: Cordycipitaceae) isolates, ACP18001 and ACP18002. Chi-square analysis of primary and secondary acquisition bioassays revealed that both field isolated strains outperformed Cordyceps (Isaria) fumosorosea (Wize) (Hypocreales: Cordycipitaceae) Apopka97 under both primary (direct spray) and secondary acquisition (adult exposure to sprayed foliage) bioassays with ACP18002 marginally outperforming ACP18001 under secondary acquisition. Slopes of the dose response regression lines for the three fungi were not significantly different. In addition, the thermal profiles for vegetative growth of each isolate indicated that the field isolates grew at higher rates than the standard at higher temperatures. The new isolates may prove to be good candidates for the management of D. citri populations in the LRGV.

Effects of Field and Greenhouse Solarization on Soil Microbiota and Weed Seeds in the Northeast USA

Soil solarization using clear plastic is a promising weed management strategy for organic farms in the Northeast USA. Based on grower concerns that the practice might negatively affect beneficial soil microbiota, we conducted experiments to measure the effects of 2 and 4 weeks of solarization in a field and a closed greenhouse. Soil microbial communities were assayed by dilution plating on semi-selective agar media. Populations of general bacteria, general fungi, bacilli, and florescent pseudomonads were unaffected by field solarization, but fluorescent pseudomonads were reduced following greenhouse solarization. At plastic removal, soil biological activity was reduced non-significantly in the field and by 45% in the green- house. Soil biological activity fluctuated following field solarization, being significantly suppressed at 5 but not 14 days after plastic removal. In the greenhouse, biological activity remained suppressed up to 28 days after plastic removal. Solarization increased available nitrogen in the field and greenhouse. Four weeks of solarization reduced viability of buried weed seeds by 64% in the field and 98% in the greenhouse, indicating that the practice can cause substantial weed seed mortality. Maximum soil temperatures, measured at 10 cm depth under solarization, were 44◦ C in the field and 50◦ C in the greenhouse; temperatures were theoretically sufficient for the reduction of some soil borne pathogens. A subsequent experiment measured the effects of solarization and tarping (black plastic) on soil biological activity. During mulching, biological activity was unaffected by treatment, but 14 days after plastic removal, biological activity was reduced in the solarized treatment as compared with the control. Overall, these results suggest that solarization can deplete the weed seedbank. Although soil biological activity was reduced by solarization, it may bounce back after a period. Greenhouse solarization achieved higher temperatures and was more lethal to weed seeds and some microbiota than field solarization.

FIRST REPORT OF BACTERIAL PITH SOFT ROT CAUSED BY PECTOBACTERIUM CAROTOVORUM subsp. BRASILIENSE ON ARTICHOKE IN ITALY

A severe soft rot disease was observed in some commercial artichoke crops (F1-hybrid cultivars) during 2014 in the Bari, Foggia (Apulia, Southern Italy) and Matera (Basilicata, Southern Italy) provinces. Symptoms were chlorosis and wilting of the older leaves accompanied by dark-green to dark-brown soft rotting of the pith. Several bacterial isolates were obtained from affected plants by culturing on semi-selective crystal violet pectate agar medium. Biochemical, physiological and molecular analyses and pathogenicity tests, performed using five selected isolates, identified three of them as Pectobacterium carotovorum subsp. brasiliense. To the best of our knowledge, this is the first report of a bacterial soft rot caused by P. carotovorum subsp. brasiliense in Italy and the first finding of this bacterial subspecies on artichoke.

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.

Prevalence and comparison of Streptococcus infantarius subsp. infantarius and Streptococcus gallolyticus subsp. macedonicus in raw and fermented dairy products from East and West Africa

Streptococcus infantarius subsp. infantarius (Sii) and Streptococcus gallolyticus subsp. macedonicus are members of the Streptococcus bovis/Streptococcus equinus complex (SBSEC) associated with human infections. SBSEC-related endocarditis was furthermore associated with rural residency in Southern Europe. SBSEC members are increasingly isolated as predominant species from fermented dairy products in Europe, Asia and Africa. African variants of Sii displayed dairy adaptations to lactose metabolism paralleling those of Streptococcus thermophilus including genome decay. In this study, the aim was to assess the prevalence of Sii and possibly other SBSEC members in dairy products of East and West Africa in order to identify their habitat, estimate their importance in dairy fermentation processes and determine geographic areas affected by this potential health risk. Presumptive SBSEC members were isolated on semi-selective M17 and SM agar media. Subsequent genotypic identification of isolates was based on rep-PCR fingerprinting and SBSEC-specific16S rRNA gene PCR assay. Detailed identification was achieved through application of novel primers enhancing the binding stringency in partial groES/groEL gene amplification and subsequent DNA sequencing. The presence of S. thermophilus-like lacS and lacZ genes in the SBSEC isolates was determined to elucidate the prevalence of this dairy adaptation. Isolates (n=754) were obtained from 72 raw and 95 fermented milk samples from Côte d'Ivoire and Kenya on semi-selective agar media. Colonies of Sii were not detected from raw milk despite high microbial titers of approximately 106CFU/mL on M17 agar medium. However, after spontaneous milk fermentation Sii was genotypically identified in 94.1% of Kenyan samples and 60.8% of Kenyan isolates. Sii prevalence in Côte d'Ivoire displayed seasonal variations in samples from 32.3% (June) to 40.0% (Dec/Jan) and isolates from 20.5% (June) to 27.7% (Dec/Jan) present at titers of 106–108CFU/mL. lacS and lacZ genes were detected in all Kenyan and 25.8% (June) to 65.4% (Dec/Jan) of Ivorian Sii isolates. Regional differences in prevalence of Sii and dairy adaptations were observed, but no clear effect of dairy animal, fermentation procedure and climate was revealed. Conclusively, the high prevalence of Sii in Kenya, Côte d'Ivoire in addition to Somalia, Sudan and Mali strongly indicates a pivotal role of Sii in traditional African dairy fermentations potentially paralleling that of typical western dairy species S. thermophilus. Putative health risks associated with the consumption of high amounts of live Sii and potential different degrees of evolutionary adaptation or ecological colonization require further epidemiologic and genomic investigations, particularly in Africa.

A SEVERE OUTBREAK OF BACTERIAL LETTUCE SOFT ROT CAUSED BY PECTOBACTERIUM CAROTOVORUM subsp. CAROTOVORUM IN APULIA (ITALY)

A severe bacterial disease was observed in lettuce plants during surveys carried out in the province of Bari (Apulia, Italy). Symptoms were chlorosis and wilting of older leaves, water-soaked and dark green soft rot of the pith. Several bacterial isolates were obtained from the affected plants by isolations on semi-selective crystal violet pectate agar medium. Biochemical, nutritional, and pathogenicity tests identified them as Pectobacterium carotovorum subsp. carotovorum. The nutritional profile obtained using the Biolog system and the 434 bp product amplified by PCR using primers designed on P. carotovorum pectate lyase-encoding gene sequence further confirmed the identity of the isolates. Some epidemiological aspects of the pathogen and the main measures to prevent and control the disease are reported.

Spatial dispersal of Metarhizium anisopliae and Beauveria bassiana in soybean fields

The dispersal and persistence of Beauveria bassiana and Metarhizium anisopliae following application in soybean fields as kaolin dust and sprays of soybean oil formulations were investigated. Fungal dispersion was evaluated from a centrally-treated area by sampling leaflets on eight transects at 20-m intervals in concentric rings up to 100 m from the central area. Density of colony-forming units (CFU) per unit leaf area was determined by washing the soybean leaflets in water containing Tween 80, and plating the aqueous suspensions on an oatmeal-dodine semi-selective agar medium. Spatial dispersal of B. bassiana in soybean field was significantly higher when conidia were applied as an oil formulation compared with a kaolin dust formulation. No significant differences were observed in M. anisopliae CFU number between sprayed or dusted plots. Oil-based formulations favor the dispersal of B. bassiana in soybean crops, but not the dispersal of M. anisopliae. Kaolin-based formulations are not suitable for B. bassiana or M. anisopliae dispersal.

Comparison of extraction procedures for detection of Xanthomonas axonopodis pv. phaseoli in common bean seed

Xanthomonas axonopodis pv. phaseoli (Xap) is an important seedborne pathogen of Phaseolus vulgaris. Accurate seed health testing methods are critical to protect seed quality and meet phytosanitary requirements. Currently employed selective media‐based methods include several variations in extraction procedures. In order to optimize pathogen extraction from seeds, the influence of different extraction steps on the sensitivity of Xap detection was assessed. Seeds were inoculated by vacuum infiltration with Xap to achieve inoculum levels from 101 to 105 CFU per seed; one contaminated seed was mixed into 1000‐seed subsamples of uncontaminated P. vulgaris seeds. Thirty subsamples of 1000 seeds were tested using each different extraction procedure. These included soaking whole seeds in sterilized saline phosphate buffer, either overnight at 4°C or for 3 h at room temperature, with or without vacuum extraction, and either with or without concentrating the seed extract by centrifuging. Seed extract dilutions were cultured on semiselective agar media MT and XCP1. The percentages of positive subsamples were compared to measure the effects of each extraction step on detection sensitivity. Vacuum extraction and centrifugation of seed extracts increased sensitivity; the highest sensitivity was obtained with the 3 h vacuum extraction followed by centrifugation. These results were confirmed with naturally infested seeds; Xap was detected in 48 of 70 samples using the 3 h vacuum extraction with centrifugation, whereas only 35 of 70 field samples tested positive using overnight soaking, a significant difference. The results suggest that these steps would be valuable modifications to the current method approved by the International Seed Testing Association (ISTA).

First Report in Burkina Faso of Xanthomonas citri pv. mangiferaeindicae Causing Bacterial Canker on Mangifera indica.

Bacterial canker of mango (or bacterial black spot) caused by Xanthomonas citri pv. mangiferaeindicae, is an economically important disease in tropical and subtropical areas (1). X. citri pv. mangiferaeindicae can cause severe infection on a wide range of mango cultivars and induces raised, angular, black leaf lesions, sometimes with a chlorotic halo. Fruit symptoms are black, star shaped, erumpent, and exude an infectious gum. A survey was conducted in Burkina Faso in May 2010 because budwood putatively associated with an outbreak of bacterial canker in Ghana had originated from Burkina Faso (3). Leaves and twigs with suspected bacterial canker lesions were collected from mango trees of the cvs. Amélie, Brooks, and Kent and from seedlings at five localities in Comoe and Houet provinces. Severe infections were observed on the sampled trees in Burkina Faso and leaf symptoms were typical of bacterial canker. Leaves were surface sterilized for 15 to 30 s with 70% ethanol, and nonpigmented, Xanthomonas-like bacterial colonies were isolated on KC semiselective agar medium (1). On the basis of an IS1595-ligation mediated PCR assay, 18 strains from Burkina Faso produced identical fingerprints and were identified as X. citri pv. mangiferaeindicae (4). The haplotype for strains from Burkina Faso was identical to that reported from Ghana (3). Three strains from Burkina Faso (LH127-2, LH130-1, and LH131-1) were compared by multilocus sequence analysis (MLSA) with the type strain of X. citri and the pathotype strain of several X. citri pathovars, including pvs. anacardii and mangiferaeindicae, targeting the atpD, dnaK, efp, and gyrB genes (2). Nucleotide sequences were 100% identical to those of the pathotype strain of X. citri pv. mangiferaeindicae, regardless of the gene assayed, but differed from any other X. citri pathovar assayed. Leaves of mango cv. Maison Rouge, taken from the youngest vegetative flush, were infiltrated (10 inoculation sites per leaf for three replicate leaves on different plants per bacterial strain) with the same three strains from Burkina Faso. Bacterial suspensions (approximately 1 × 105 CFU/ml) were prepared in 10 mM Tris buffer (pH 7.2) from 16-h-old solid cultures on YPG agar (7 g of yeast, 7 g of peptone, 7 g of glucose, and 18 g of agar per liter, pH 7.2). The negative control treatment consisted of three leaves infiltrated with sterile Tris buffer (10 sites per leaf). Plants were incubated in a growth chamber at 30 ± 1°C by day and 26 ± 1°C by night (12-h/12-h day/night cycle) at 80 ± 5% relative humidity. Typical symptoms of bacterial canker were observed for all assayed strains 1 week after inoculation; no symptoms were observed from negative control leaves. One month after inoculation, mean X. citri pv. mangiferaeindicae populations ranging from 2 × 107 to 8 × 107 CFU/leaf lesion were recovered, which was typical of a compatible interaction (1). The origin of inoculum associated with the bacterial canker outbreak in Burkina Faso is unknown. This report documents severe infections in Burkina Faso (including premature fruit drop due to severe fruit infections) and confirms the presence of bacterial canker in western Africa. A more extensive survey for the disease should be conducted in this region.

First Report in Ghana of Xanthomonas citri pv. mangiferaeindicae Causing Mango Bacterial Canker on Mangifera indica.

Bacterial canker of mango (or bacterial black spot), caused by Xanthomonas citri pv. mangiferaeindicae, is an economically important disease in tropical and subtropical producing areas (1). X. citri pv. mangiferaeindicae can cause severe infection in a wide range of mango cultivars and induces raised, angular, black leaf lesions, sometimes with a chlorotic halo. Several months after infection, leaf lesions dry and turn light brown or ash gray. Severe leaf infection may result in abscission. Fruit symptoms appear as small water-soaked spots on the lenticels. These spots later become star shaped, erumpent, and exude an infectious gum. Often, a "tear stain" infection pattern is observed on the fruit. Severe fruit infections will cause premature fruit drop. Twig cankers are potential sources of inoculum and weaken resistance of branches to wind damage. Leaf lesions with suspected bacterial canker were collected in January 2010 from mango trees cv. Keitt in several blocks at the Integrated Tamale Fruit Company, Ghana. Non-pigmented Xanthomonas-like bacterial colonies were isolated on Kasugamycin-Cephalexin semiselective agar medium (3). On the basis of IS1595-Ligation Mediated-PCR data, 16 strains from Ghana produced identical fingerprints and were identified as X. citri pv. mangiferaeindicae (4). The haplotype corresponding to the Ghanaian strains had not been previously reported. On the basis of multidimensional scaling (4), this haplotype clustered together with a group of strains from multiple origins and the analysis was not informative as an aid for tracing back the outbreak. Five Ghanaian strains (LH2-3, LH2-6, LH2-8, LH2-11, and LH2-15) were compared by multilocus sequence analysis to the type strain of X. citri and the pathotype strain of several X. citri pathovars, including pvs. anacardii and mangiferaeindicae. This assay targeted the atpD, dnaK, efp, and gyrB genes as described previously (2). Nucleotide sequences were 100% identical to those of the pathotype strain of X. citri pv. mangiferaeindicae whatever the gene assayed, but differed from any other assayed X. citri pathovar. Mango cv. Maison Rouge leaves from the youngest vegetative flush were infiltrated (10 inoculation sites per leaf, three replicate plants) using inoculum of each of the same five Ghanaian strains made from suspensions in Tris buffer containing ~1 × 105 CFU/ml. Negative control treatments consisted of leaves infiltrated with sterile Tris buffer. Typical symptoms of bacterial canker were observed for all assayed strains a week after inoculation. No lesions were recorded from the negative control. One month after inoculation, mean X. citri pv. mangiferaeindicae population sizes ranging from 4 × 107 to 1 × 108 CFU/lesion were recovered from leaf lesions, typical of a compatible interaction (1). High disease prevalence was observed in Ghana, indicating the suitability of environmental conditions in this region for the development of mango bacterial canker. The budwood for these blocks was imported from Burkina Faso in 2002 and symptoms were observed in these blocks shortly after establishment. To our knowledge, this is the first report of mango bacterial canker in Western Africa.

A New Type of Strain of Xanthomonas euvesicatoria Causing Bacterial Spot of Tomato and Pepper in Grenada.

Bacterial spot of tomato and pepper (BSTP) can be caused by several Xanthomonas genospecies (2). BSTP is a major disease in Grenada where A and B phenotypic groups (Xanthomonas euvesicatoria and X. vesicatoria, respectively, [2]) have been reported (3). There is no previous report of group A strains, which are strongly amylolytic and pectolytic, in Grenada. In March 2007, tomato and pepper leaves with lesions typical of BSTP were collected in Saint David and Saint Andrew parishes of Grenada. Bacterial isolations were performed on KC semiselective agar medium (4), resulting in isolation of five yellow-pigmented, Xanthomonas-like strains. Three strains isolated from tomato or pepper in Saint David were negative for starch hydrolysis and pectate degradation, two tests that were found useful for strain identification in the 1990s (2). Two strains isolated from pepper in Saint David were strongly amylolytic and degraded pectate. Amplified fragment length polymorphism (AFLP) and multilocus sequence analysis (MLSA) assays targeting atpD, dnaK, efp, and gyrB were performed on the five strains from Grenada together with a type strain of each of X. euvesicatoria, X. perforans, X. gardneri, and X. vesicatoria as well as other reference strains of X. euvesicatoria and X. perforans as described previously (1). All strains from Grenada were identified as X. euvesicatoria regardless of the typing technique. On the basis of AFLP assays, the two strains with phenotypic features not reported in Grenada were closely related (distances of ≤0.002 nucleotide substitutions per site [1]) to a group of strains from India (ICMP 3381, LMG 907, LMG 908, and LMG 918). These two strains were also identical to the Indian strains based on MLSA, but differed from the X. euvesicatoria type strain by at least one nucleotide substitution in all loci examined. The three strains from Grenada that were negative for starch hydrolysis and pectate degradation had sequences identical to that of the type strain. Young leaves of tomato plants of cv. Marmande and pepper plants of cvs. Yolo Wonder and Aiguille were infiltrated (six inoculation sites per leaf, three replicate plants per cultivar per experiment, and the experiment was replicated once) using inoculum of each of the five strains from Grenada made from suspensions in Tris buffer containing approximately 1 × 105 CFU/ml. Two reference strains of X. euvesicatoria (NCPPB 2968 and LMG 922) were also inoculated as positive control treatments. Negative control treatments consisted of leaves infiltrated with sterile Tris buffer. Typical water-soaked lesions that developed into necrotic spots were observed 3 to 8 days after inoculation (dai) for all strains on all cultivars, except NCPPB 2968, which was not pathogenic on pepper cv. Aiguille. Xanthomonas population sizes from lesions plated onto KC agar medium (4) 25 dai ranged from 3 × 106 to 5 × 107, 8 × 107 to 2 × 108, and 9 × 106 to 2 × 108 CFU/lesion on tomato cv. Marmande and pepper cvs. Yolo Wonder and Aiguille, respectively. The epidemiological importance of this previously unreported group of X. euvesicatoria strains in Grenada needs to be assessed.

Recent Outbreaks of Black Rot of Brassicas Caused by Xanthomonas campestris pv. campestris in Southern Mozambique.

In August and September of 2007, black rot symptoms were observed on seedbed and field plants of Brassica spp. grown in the southern districts of Boane, Mahotas, and Chòkwé in Mozambique. One hundred eighty-two cabbage-growing households were evaluated for the incidence of Xanthomonas campestris pv. campestris. Five Brassica cultivars, Glory F1, Glory of Enkhuizen, Copenhagen Market, Starke (Brassica oleracea pv. capitata L.), and Tronchuda (B. oleracea L. var. costata DC) were grown in the areas for several years. The hybrid Glory F1 was the most popular grown cultivar in the surveyed areas. In the Boane district, the highest incidence of black rot was recorded on Copenhagen Market (70%), Starke (67.9%), and Glory F1 (67.3%). In Chòkwé, Tronchuda (Portuguese kale) was the least affected Brassica crop. Water-soaked lesions starting at the edge of leaves with typical V-shaped necrotic lesions and vein discoloration were the most commonly observed symptoms. When examined with a microscope, cut edges of symptomatic stem and leaf tissues consistently exhibited bacterial streaming. The bacteria were isolated from commercial seed and field-grown plants on semiselective agar media (2). Forty-six X. campestris pv. campestris strains that were gram negative, aerobic, starch positive, nitrate negative, and oxidase negative or weakly positive (3) were further identified on the basis of ELISA (Agdia Inc., Elhart, IN), GN Biolog Microbial Identification System, version 4.2 (Biolog Inc., Hayward, CA), and PCR-specific primers (1). Pathogenicity tests were conducted by pin inoculating two upper leaves of cabbage (cv. Wirosa) in the 2- to 3-leaf stage with bacterial growth from 24-h-old agar cultures (2). Black rot symptoms developed on nearly all inoculated plants within 7 to 14 days. No symptoms were observed on control plants inoculated with a sterile pin without bacterial inoculum. The severity of black rot of Brassica spp. in three important farming districts caused significant losses in Mozambique.

The epidemiological significance of post-packinghouse survival of Xanthomonas citri subsp. citri for dissemination of Asiatic citrus canker via infected fruit

The risk of introduction of Xanthomonas citri subsp. citri ( Xcc) to new, unaffected citrus producing areas is a major concern for those citrus industries attempting to remain free of citrus canker. Citrus fruit, as a potential pathway for Xcc to enter and become established in these areas, are assumed to be a risk. However, there is little information relative to the potential of harvested fruit to act as an inoculum source. A multi-national research team was established to investigate the potential of bacterial survival in infected citrus fruit lesions and as surface contaminants on symptom-free fruit, and to examine the potential of infected fruit as a viable inoculum source. Experiments were conducted in various locations in Florida and Argentina. Bacterial recovery and culture plating were problematic due to the presence of non-pathogenic bacteria with cultural characteristics that were difficult to distinguish from Xcc. Therefore, in all experiments, although culturing on semi-selective agar media was used as an indication of overall bacterial populations, bioassays were conducted via needleless injection and infiltration of suspect bacterial suspensions into susceptible cv. Duncan grapefruit leaves. Inoculation sites were subsequently assessed for symptoms of citrus canker and lesions were individually enumerated to confirm the presence of Xcc. In commercial packing lines in Florida and northwest Argentina, prewashing the fruit to remove dirt and debris reduced surface bacterial populations. As anticipated, recovery of Xcc from fruit surfaces increased when active citrus canker lesions were present but total bacterial recovery decreased after processing, and bioassays demonstrated that the quantity of viable Xcc declined as fruit remained in cold storage, or as they aged on the trees. Bioassays demonstrated that the highest incidence of Xcc from fruit after the packing line antimicrobial treatment occurred with symptomatic fruit (2.5–50.6 lesions leaf −1), and zero to very low levels with fruit from apparently healthy trees (0–1.74 lesions leaf −1). Furthermore, the proportion of injection–infiltration bioassay sites that developed lesions consistently decreased with time after processing in each of the three packinghouse studies, also showing that as fruit senesce and lesions age the ability of fruit to generate or sustain Xcc bacteria was increasingly compromised. The packing line process reduced canker lesion activity by as much as 50% compared to unprocessed fruit. Xcc survived in wounds on mature fruit attached to the tree, but Xcc populations declined in wounds of processed or non-processed harvested fruit. Discarded canker-infected fruit in cull piles was ineffective as a source of inoculum for dispersal. Transmission from cull piles of packing line-processed fruit to surrounding trap plants, even less than 1 m away, did not occur under natural conditions. However, with severely infected piles of culled fruit subject to extreme simulated wind (25 m s −1) and rain conditions, only a single lesion, associated with leaf injury, developed on a trap plant immediately downwind of the cull pile, suggesting an exceedingly low risk of spread. Taken as a group, this series of experiments demonstrate that harvested and packinghouse-disinfested citrus fruit are extremely unlikely to be a pathway for Xcc to reach and infect susceptible citrus and become established in canker-free areas.

Quantification of Fusarium solani f. sp. glycines isolates in soybean roots by colony-forming unit assays and real-time quantitative PCR

Fusarium solani f. sp. glycines (FSG; syn. F. virguliforme Akoi, O'Donnell, Homma & Lattanzi) is a soil-borne fungus that infects soybean roots and causes sudden death syndrome (SDS), a widespread and destructive soybean disease. The goal of this study was to develop and use a real-time quantitative polymerase chain reaction (QPCR) assay to compare the accumulation of genomic DNA among 30 FSG isolates in inoculated soybean roots. Isolates differed significantly (P < or = 0.05) in their DNA accumulation on a susceptible soybean cultivar when detected and quantified using a FSG-specific probe/primers set derived from the sequences of the nuclear-encoded, mitochondrial small subunit ribosomal RNA gene. QPCR results that were normalized as the fold change over the sample collection times after inoculation were significantly (P < or = 0.001) correlated with the log(10) transformed colony-forming unit (CFU) values of FSG obtained from plating of inoculated ground roots on FSG semi-selective agar medium. Several isolates were identified that accumulated more FSG DNA and had higher CFU values than the reference isolate FSG1 (Mont-1). Compared to other isolates, FSG5 was the most aggressive root colonizer based on DNA accumulation and CFU values in infested roots. The described QPCR assay should provide more specificity, greater sensitivity, and less variability than alternatives to the culturing-dependent and time-consuming plating assays. Evaluation of isolate relative DNA differences on host plants using the QPCR approach provides useful information for evaluating isolates based on the extent and/or degree of colonization on soybean roots and for selecting isolates for breeding SDS-resistant soybean lines.

Rusted Root of Ginseng (Panax quinquefolius) Is Caused by a Species of Rhexocercosporidium

ABSTRACT Rusted root (also known as rusty root) of ginseng (Panax quinquefolius) was first described over 70 years ago, but the causal agent has not been clearly established. The disease is characterized by slightly raised reddish-brown to black root lesions of varying size. The lesions, regardless of size, remain superficial; however, peridermal tissue is ruptured and sloughed off, giving the root a scabbed appearance. Culture-independent techniques were used to demonstrate that a fungal internal transcribed spacer (ITS) region DNA fragment was strongly associated with diseased but not healthy root tissue. The fragment ( approximately 650 bp in length) was cloned. Restriction enzyme digests of cloned DNA indicated that the 650-bp fragment represented a single taxon. BLAST analysis following sequencing of the fragment found that the nearest matches in GenBank were anamorphic genera associated with discomycetes, in particular Rhexocercosporidium spp. This putative identification was supported further by isolating fungi from diseased tissue using a semiselective agar medium. With this procedure, a Rhexocercosporidium-like fungus was isolated; DNA extracted from fungal cultures and amplified using ITS oligonucleotide primers was found to be identical to similarly amplified DNA from the 650-bp bands. However, the isolates were distinct, with respect to growth rate on agar media and ITS sequence, from Rhexocercosporidium carotae, the only described species in this genus. The ability to reproduce symptoms on ginseng roots was confirmed in pathogenicity tests. Oligonucleotide primers based on ITS sequences were designed to amplify DNA of Rhexocercosporidium spp. Polymerase chain reaction assays on DNA extracted from naturally infected root tissue showed that the fungus was present in nearly all symptomatic roots but was infrequent in healthy-appearing roots. The most probable cause of rusted root of ginseng is a previously undescribed species of Rhexocercosporidium.

A Semi-Selective agar medium to detect the presence of Xanthomonas axonopodis pv. malvacearum in naturally infected cotton seed

A semi-selective agar medium was developed for detection of Xanthomonas axonopodis pv. malvacearum (Xam) in cotton (Gossypium hirsutum) seed. The basic medium was peptone-sucrose-agar (PSA). Criteria for the semi-selective medium were the typical colony characters of Xam and its pathogenicity on cotton. Several systemic fungicides and antibiotics in different concentrations were tested alone or in combination with others. The final composition of the semi-selective agar medium was established after several attempts in order to inhibit most of the fungal and bacterial saprophytes and favour the development of Xam. It contained PSA + cyclohexamide, cephalexin, pencycuron, triadimenol and tolylfluanid. The bacteria were recovered from naturally infected seeds by the direct plating of 2,000 surface disinfected seeds on the semi-selective medium. The recovery of the pathogen from naturally infected leaf tissues and in dilution plating, on semi-selective medium and on nutrient agar, were comparable. Among the three detection methods tested, the semi-selective medium was found to be the most reliable and quantifiable. Degree of severity of angular leaf spot in the field was not always correlated with the level of infection in the seed. This is the first report of a semi-selective agar medium to detect the presence of Xam in naturally infected cotton seed.

Epiphytic survival of Pseudomonas viridiflava, causal agent of pith necrosis of tomato, on weeds in Turkey.

Pith necrosis, caused by Pseudomonas viridiflava (Burkholder) Dowson, has been an important problem of greenhouse-grown tomatoes in the eastern Mediterranean region of Turkey since 1998. In the study, the epiphytic survival of P. viridiflava on weeds was investigated by using a semi selective agar medium (T-5) and indirect-ELISA in commercial greenhouses with a history of pith necrosis in three different locations of the region during 2001 and 2002. It is the first study showing that pathogenic strains of P. viridiflava on tomato may survive as an epiphytic population on the phyllosphere of Amaranthus spp., Conyza canadensis, and Orobanche ramosa in Turkey. The strains were identified as P. viridiflava by morphological, physiological, biochemical tests, indirect-ELISA and fatty acid methyl ester analysis, and were found to be pathogenic on tomato plants.

Morphology of Verticillium dahliae and V. tricorpus on semi-selective media used for the detection of V. dahliae in soil

The morphology of two soil-borne Verticillium species, V. dahliae and V. tricorpus, was studied on two semi-selective agar media, in the absence and presence of soil. Morphology of the fungi differed considerably between the media, with respect to presence and shape of microsclerotia, dark hyphae (i.e. short melanised hyphae attached to the microsclerotia) and dark mycelium (i.e. melanised mycelium throughout the colony). On modified soil extract agar (MSEA), a pectate based agar, V. dahliae always had globose to elongate microsclerotia, without dark hyphae or dark mycelium, whereas V. tricorpus always had dark hyphae or dark mycelium, and microsclerotia, whenever present, were globose to irregular in shape. On ethanol agar (EA), V. dahliae had large microsclerotia and abundant dark hyphae, whereas V. tricorpus did not form microsclerotia, but always abundant dark mycelium. For the first time we observed the formation of dark hyphae by V. dahliae to a great extent. In the presence of soil, most characteristics were less pronounced, and V. dahliae microsclerotia were smaller, but V. tricorpus produced large microsclerotia, even when they were absent in pure culture. Morphological characteristics suitable for discrimination between the two species on MSEA plates in the presence of soil were selected and tested with fresh isolates from agricultural fields. The two fungi could be distinguished using qualitative characteristics and microsclerotial size. Molecular analysis and morphology on potato dextrose agar confirmed all identifications made on soil dilution plates.

Survival of Clavibacter michiganensis ssp. michiganensis in infected tomato stems under natural field conditions in California, Ohio and Morocco

The survival and half‐life of Clavibacter michiganensis ssp. michiganensis (C. michiganensis), the causal agent of bacterial canker of tomato, were determined in infected plant debris under natural field conditions in California, Ohio and Morocco using a semiselective agar medium. The organism survived significantly longer in tomato stems left on the soil surface than in stems buried in the soil at all locations studied. The pathogen was recovered in high amounts from tomato stems left on the soil surface for 314 days in Ohio and California, USA, and for 194 and 132 days in Melk Zhar and Aït Melloul, Morocco, respectively; it was recovered from stems buried in the soil for up to 314 days in Ohio, up to 240 days in California, and up to 60 days in Aït Melloul and Melk Zhar. The half‐life of the pathogen in stems left on the soil surface ranged from 23·2 to 24·8 days in the USA, and from 7·8 to 12·3 days in Morocco, whereas the half‐life in buried stems ranged from 14·0 to 16·7 days in the USA and from 3·7 to 9·5 days in Morocco. Based on the half‐life data, the predicted survival times of C. michiganensis in stems on the soil surface in Ohio, California, Melk Zhar and Aït Melloul would be up to 822, 770, 424 and 261 days, respectively, while the predicted survival times in stems buried in the soil would be 541, 497, 305 and 128 days, respectively. These results show that the survival and half‐life of C. michiganensis in plant debris are relatively long and are influenced by both tissue exposure and geographic location.

Development of a New Semiselective Medium for Isolating Xanthomonas campestris pv. manihotis from Plant Material and Soil

ABSTRACT An effective control for bacterial blight of cassava (Manihot esculenta), caused by Xanthomonas campestris pv. manihotis, requires the use of non-contaminated cuttings and seeds. Using classical agar plating techniques for screening planting material for contamination has not been very successful because of the lack of a reliable semiselective agar medium. The pathogen grows slowly on general plating media and is easily overgrown by saprophytic bacteria during isolation from diseased plants. In an effort to develop a semiselective medium, the utilization of several carbon and nitrogen sources was studied. Results of these tests provided information used to design a basal medium allowing good growth of the target organism while suppressing growth of several common saprophytes. Additional selectivity was achieved by incorporating three antibiotics into the basal medium. The new semiselective agar medium, designated cefazolin trehalose agar (CTA) medium, contained (per liter) 3.0 g of K(2)HPO(4), 1.0 g of NaH(2)PO(4), 0.3 g of MgSO(4).7H(2)O, 1.0 g of NH(4)Cl, 9.0 g of D(+)-trehalose, 1.0 D(+)-glucose, 1.0 g of yeast extract, 0.025 g of cefazolin, 0.0012 g of lincomycin, 0.0025 g of phosphomycin, 0.25 g of cycloheximide, and 14.0 g of agar. In comparison to a starch-based semiselective medium (SXM), plating efficiencies using pure cultures of 10 strains of X. campestris pv. manihotis were significantly higher on CTA, with an average of 85 and 50%, respectively. Likewise, isolation and recovery of X. campestris pv. manihotis from infected cassava leaves and contaminated soil were much higher on CTA than on SXM agar. When X. campestris pv. manihotis occurs in high concentrations in diseased tissue, the standard yeast trehalose glucose agar medium supplemented with 250 mug of cycloheximide per ml appears to be satisfactory. The newly developed CTA medium should prove useful for control strategies to identify and remove infected planting material of cassava, as well as for basic ecological studies of the pathogen.