Specific detection of Ralstonia solanacearum 16S rRNA sequences by AmpliDet RNA

The potential of AmpliDet RNA for specific detection of Ralstonia solanacearum in potato tuber samples and surface water was demonstrated. AmpliDet RNA is a procedure based on nucleic acid sequence based amplification (NASBA) of RNA sequences and homogeneous real time detection of NASBA amplicons with a molecular beacon. The procedure is carried out in sealed tubes, thus reducing the risks for carry-over contamination. AmpliDet RNA enabled reliable detection of specific 16S rRNA sequences of R. solanacearum in total RNA extracts from potato tuber samples in 90min at a level of 10 cells per reaction, equivalent to ca. 10⁴cellsml^–1 of sample. In surface water, AmpliDet RNA allowed detection of R. solanacearum at a level of 10cfuml^–1, after concentrating bacteria from 200ml of surface water into 1ml of surface water by centrifugation.All strains of R. solanacearum and a strain of R. syzygii were positive in AmpliDet RNA, but not other (related) bacterial species. Ralstonia solanacearum (race 3, biovar 2) could be detected reliably in 18 naturally infected potato tuber samples containing varying concentrations of cells. Ninety-one negative tuber samples, from which no R. solanacearum was isolated, were tested in AmpliDet RNA, including 23 samples containing bacteria (cross-) reacting with antibodies against R. solanacearum in immunofluorescence (IF) cell-staining. Only one negative sample, containing high numbers of IF-positive cells, was positive in AmpliDet RNA.     

Influence of mixed biocide GCSC-BtA on the pupae and adult stages of Apanteles plutellae Kurd. (Hym., Braconidae) and its host. Plutella xylostella (L.) (Lep., Plutellidae)

This paper deals with the influence of the mixed biocide GCSC-BtA on the pupal and adult stages of Apanteles plutellae Kurd. (Hym., Braconidae) and its host, Plutella xylostella (L.) (Lep., Plutellidae). The results show that mortalities of the pupae of P. xylostella in the direct-dip bioassay were 84.67%, that of the adults in the residue bioassay at 1.2500mg/ml concentration of GCSC-BtA were 78.00% which were significantly higher than the mortality values for the pupae with 54.62% and adults with 48.13% of A. plutellae. In contrast, cypermethrin showed extremely high toxicity to the pupae with 94.58% and adults with 86.00% mortality values of A. plutellae as compared to the low mortality values of 42.14% for the pupae and 32.11% for the adults of P. xylostella, with the same concentrations and bioassay methods. The LC₅₀ values of GCSC-BtA were 0.3402, 0.5516 and 1.2405, 1.9480mg/ml for the pupae and adults of P. xylostella and A. plutellae, respectively, while the LC₅₀ values for cypermethrin were 1.5652, 2.3471 and 0.1096, 0.1152mg/ml, respectively. GCSC-BtA was found more toxic to the pupae and adults of P. xylostella and safer to the pupae and adults of A. plutellae than cypermethrin. The possibilty of using GCSC-BtA against P. xylostella under partial control by A. plutellae in vegetable fields is discussed.     

Investigations on side-effects of the mixed biocide GCSC-BtA on different predators of Plutella xylostella (L.) (Lep., Plutellidae) in southeastern China

The present paper deals with laboratory studies on side-effects of the mixed biocide GCSC-BtA on Plutella xylostella (L.) (Lep., Plutellidae) and its predators, e.g. Amblyseius longispinosus (Evans) (Acari, Phytoseiidae), Erigonidium graminicola (Sundvall) (Araneae, Linyphiidae), Orius similis Zheng (Het., Anthocoridae) and Coccinella septempunctata L.(Col., Coccinellidae), in comparison to the commercial insecticides, e.g. Abamectin, Tebufenozide, Dichlorvos, Cartap and Lambda-cyhalothrin.The results showed that GCSC-BtA was highly toxic to the 3^rd instar of P. xylostella with 91.18% mortality, followed by Cartap with 84.38%, Abamectin with 78.00%, Tebufenozide with 75.57%, Lambda-cyhalothrin with 63.75% and Dichlorvos with 50.86% mortality. On the other hand, GCSC-BtA was found to be comparatively less toxic to the predators, causing 31.11%, 13.33%, 11.54% and 6.00% mortalities in A. longispinosus, E. graminicola, O. similis and C. septempunctata, respectively. For comparison, the mortalities recorded for Abamectin, Tebufenozide, Dichlorvos, Cartap, Lambda-cyhalothrin were 72.94%, 55.55%, 70.00%, 53.26% and 98.85% in A. longispinosus, 46.51%, 55.10%, 60.00%, 46.00% and 73.68% in E. graminicola, 22.00%, 16.00%, 35.71%, 26.78% and 81.03% in O. similis, 15.55%, 19.64%, 28.00%, 16.66% and 41.79% in C. septempunctata, respectively.Cluster analysis was introduced to group the mortalities caused by the treatments into three toxicity groups with the distance of D=4.1. The 1^st group consisted of GCSC-BtA characterized with low toxicity to all the predators tested with 15.49% mortality on average (highest 31.11% and lowest 6.00%). The 2^nd group consisted of Abamectin, Tebufenozide, Dichlorvos and Cartap with moderate toxicity to the predators with 39.96% mortality on average. The 3^rd group included Lambda-cyhalothrin with high toxicity to the predators with 73.83% mortality on average (highest 98.85% and lowest 41.70%). The susceptibility of the pest and its predators to GCSC-BtA and the insecticides is discussed. GCSC-BtA as a biological control agent is recommended for use in the integrated pests control programs in the vegetable fields.     

Suppressive effect of potassium silicate on powdery mildew of strawberry in hydroponics

From 1996 to 1997, potassium silicate (SiO₂) was tested at 0, 25, 50, and 100mgl^–1 in hydroponics to control powdery mildew. Other elements were added in the usual amounts, and the strawberries were cultivated hydroponically in a greenhouse for 4 months (from October to January). The powdery mildew spread in the control plot, but little mildew developed in the plot with 25mgl^–1 silicate, and none in plots with more than 50mgl^–1 silicate. The suppressive effect lasted for about 4 months on fruits and even longer on leaves. On analysis of mineral content in the leaves, only the silicate content differed markedly between the control and treated plants. Nitrogen, phosphate, potassium, and calcium contents did not differ greatly. The maximum silicate content was about 24 times that of the control, and disease severity decreased significantly when the content was more than 1.5% in the leaves. The hardness of the strawberry leaves, measured by a creep meter, was increased by the silicate treatment.     

Conjugation of δ-endotoxin from Bacillus thuringiensis with abamectin of Streptomyces avermitilis as a new type of biocide, GCSC-BtA, for control of agricultural insect pests

Conjugation of -endotoxin from Bacillus thuringiensis with abamectin, a toxin of Streptomyces avermitilis, was carried out to form a new type of biocide, GCSC-BtA based on Germany-China Scientific Cooperation research, for the control of agricultural insect pests. The strategy for biochemical linkage was designed by conjugating an amino group in B.t. protoxin with a carboxyl group in carboxylated abamectin under the treatment of conjugator EDC [1-Ethyl-3-(3-dimethylaminopropyl carbodiimide Hydrochloride)]. The formation of B.t. protoxin was processed by solubilizing B.t. crystal in 25mM dithiothreitol (DTT) at 37°C for 2h. The carboxylated abamectin was formed by carboxylating the NaH-activated abamectin with 10mg/ml butyric anhydride at 111°C in a water-circumfluent condensation device for 2h. The conjugating reaction, consisting of 5mg/ml B.t. protoxin, 10mg/ml carboxylated abamectin and 19.17mg/ml EDC, was successfully conducted at room temperature for 24h. Significant differences were found between pure abamectin, carboxylated abamectin and the conjugated BtA by means of UV-photo absorptions recorded at wavelengths 354, 438, 518, 600nm (P<0.01). LT₅₀ of the conjugated GCSC-BtA to the 3^rd instar larvae of Plutella xylostella (L.) (Lep., Plutellidae) was 35.27 g a.i./ml, about 62% and 76% of that caused by the B.t. protoxin and the caxboxylated abamectin, respectively. The conjugated GCSC-BtA caused 87.14% mortalities in larvae of P. xylostella, 93.75% in adult Myzus persicae (Sulzer) (Hom., Aphididae) and 89.33% in adult Phyllotreta vittata Fabricius (Col., Chrysomelidae) as compared to 48.33% by the B.t. crystal only in P. xylostella. The symptoms caused by conjugated GCSC-BtA in the 3^rd instar of P. xylostella were black color in the head part and white-yellow in the abdomen of dead larvae, which differed from the black color or the white-yellow all along the body caused by either the B.t. crystal or the abamectin, respectively. It was concluded that the conjugated GCSC-BtA biocide had a broader host spectrum and a faster killing speed than either the B.t. crystal or abamectin alone for the control of agricultural pests.     

Gamma radiation sensitivity of larvae and adults of the red flour beetle, Tribolium castaneum Herbst

In this study, the susceptibility of larvae and adults of Tribolium castaneum (Herbst) to gamma radiation was investigated in the laboratory in Turkey. Gamma radiation was applied at 6 dose levels between 20 and 200Gy to 13–15day-old adults and at 5 dose levels between 20 and 180Gy to 18–20day-old larvae of T. castaneum. All experiments were performed in growth chambers maintained at 27±1°C and 70±5% r.h. The larvae proved to be the more susceptible stage, survival to the adult stage being prevented by exposure of the eggs to 100Gy; the adult stage was less susceptible. LD₅₀ and LD₉₉ values were determined as 19,75 and 42,97Gy for larvae and 33,21 and 64,50Gy for adult stage, respectively. It was concluded that 100Gy is the effective dose for both larval and adult stages.     

Biological characteristics and life tables of Neoseiulus umbraticusChant (Acari, Phytoseiidae) at three constant temperatures

The development time, survival and fecundity of the generalist predatory mite, Neoseiulus umbraticusChant, were determined at 20, 25, and 30°C and 65±10% RH. N. umbraticus females completed development in 9.7, 8.0 and 5.9 days, respectively, using a diet of all life stages of Tetranychus cinnabarinus Boisduval. Total developmental times of males were relatively shorter at 25 and 30°C than at 20°C. In general, preoviposition, oviposition, and postoviposition periods of N. umbraticus shortened as temperature increased. The longest survival rate of N. umbraticus of 80.5 days occurred at 20°C, followed by 67.0 and 57.6 days at 25 and 30°C, respectively. Mated females laid an average 0.9, 1.3 and 1.4 eggs per female per day and 33.1, 44.0 and 43.6 eggs over their entire lives at 20, 25 and 30°C, respectively. The sex ratios of this species were 0.57, 0.57 and 0.54 female (female+male) at 20, 25 and 30°C, respectively. The intrinsic rate of increase (r_m) became greater with rising temperatures from 0.123 at 20°C to 0.180 at 30°C. The net reproduction rate (R_o) was highest at 25°C (25.0 females/female) and lowest at 20°C (18.8 females/female), while T_o decreased with increasing temperatures, from 23.8 days at 20°C to 17.5 days at 30°C.     

Phytogard® and DL-β-amino Butyric Acid (BABA) Induce Resistance to Downy Mildew (Bremia Lactucae) in Lettuce (Lactuca sativa L)

Downy mildew of lettuce (Bremia lactucae) is a serious disease. An alternative to chemicals is the application of disease resistance inducers. The aim of this study was to test whether DL--amino butyric acid (BABA) and Phytogard® (K₂HPO₃) could induce resistance in downy mildew susceptible plants. Aqueous solutions of BABA (0, 10, 20, 30, 50, 80, 100mM) and Phytogard® (0.0, 5.8, 29.0, 40.6, 58.0 and 87.0ppm) were sprayed on seven-day-old seedlings 0, 3, 7 and 15 days before or 1–3 days after inoculation with B. lactucae. Results obtained showed that Phytogard®- and BABA-induced resistance was dose-dependent. At 40.6ppm for Phytogard® and 10mM for BABA, complete protection was obtained. Both compounds had a curative effect and the induced resistance lasted for at least 15 days. It was also shown that both compounds induced systemic resistance in lettuce against downy mildew. Phytogard® at 40.6ppm completely inhibited spore germination while BABA at 20mM did not. Pathogenesis related (PR) protein analysis showed that BABA induced weak accumulation of PR-2, but not PR-1, PR-5 and PR-9. Phytogard® induced none of these proteins. The use of these two compounds to protect lettuce from B. lactucae is discussed.     

Effects of soil solarization on nematodes in Croatia

For the first time soil solarization was investigated in Croatia both in the field and in the greenhouse in 1991, 1992, 1993 and 1994. For two months (July and August), the soil was mulched with transparent polyethylene (PE) sheets of 0.015 or 0.050mm thickness. Soil temperatures at depths of 5, 10 and 20cm were recorded daily. In order to assess nematode population densities, soil samples were analysed before mulching and at the end of the mulching treatment. The results of these experiments showed that soil solarization drastically reduced the population of plant-parasitic nematodes (Meloidogyne, Pratylenchus, Paratylenchus, Tylenchus, Tylenchorhynchus spp.) by about 97–100% at a depth of 10cm and 92–97% at a depth of 20cm in the field, while in the greenhouse, the population of plant-parasitic nematodes was reduced by about 89–100% at a depth of 10cm and 98–100% at a depth of 20cm.In the same experiments, the population of saprophytic nematodes in the field was reduced by about 86–90% at a depth of 10cm and 72–89% at a depth of 20cm. In the greenhouse, the population of saprophytic nematodes was reduced by about 87–97% at a depth of 10cm and 87–93% at a depth of 20cm. This data shows that soil solarization was less effective in the control of saprophytic nematodes, which is considered to be an advantage.     

Effect of Ultraviolet Disinfection of Hydroponic Solutions on Pythium Root Rot and Non-target Bacteria

Dispersal of root pathogens is a major concern in closed hydroponic cultures. To limit dispersal, ultraviolet (UV) disinfection technology has been used to remove pathogens but its effect on non-target bacterial populations is largely unknown. In this study, the circulating solution was disinfected with various UV doses (i.e., 19, 38, 59, 88mJcm^–2) before recirculation. At the beginning of the experiment, the hydroponic solution was inoculated with Pythium aphanidermatum at 6.7±1.5CFUmL^–1. Four months later the population density of P. aphanidermatum reached 1030CFUmL^–1 in the control and 1028, 970, 610, and 521CFUmL^–1 in the solutions treated by the above UV doses. In all UV-treated solutions, significant (P<0.05) reduction of both Pythium and bacterial populations was noted. For the former, its reduction did not result in decrease of Pythium root rot nor increase of tomato fruit yield. For the latter, its reduction showed a concomitant decrease of the populations in the rhizosphere. The bacterial populations in the rhizosphere were 6.77×10⁹CFUmg^–1 fresh roots at the beginning of the experiment and were 7.89×10⁸, 9.93×10⁷, 7.33×10⁷, and 3.51×10⁷CFUmg^–1 fresh roots at the end of the experiment in the control, UV38, UV59, and UV88 treatments, respectively. The bacterial density also decreased with time in the control (UV0) although at a low rate. The results suggest that the attempt to control Pythium root rot by UV irradiation of recirculating solutions to remove P. aphanidermatum also affects the non-target bacterial populations in the rhizosphere. The interaction between the target pathogen and non-target bacterial flora in UV-treated hydroponics needs further investigation.     

Passive dispersal of the grape rust mite Calepitrimerus vitis Nalepa 1905 (Acari, Eriophyoidea) in vineyards

Modes of passive dispersal of the grape rust mite Calepitrimerus vitis (Eriophyoidea) were investigated in a vineyard of South-Western Germany. More than 200 Eriophyoidea per month were trapped in a wind chamber during summer (32,1% C.vitis) suggesting long-distance dispersal by air currents. Rain washed part of the adult C.vitis population from the foliage. SE micrographs suggest that quiescent nymphs are affixed to the leaf by a substance of unknown nature. However, the role of rain in C.vitis colonisation of uninfested vineyards is still unclear, as is the role of phoretic transport by arthropods. For the first time, evidence of rust mite dispersal by human activity is presented. A large number of C.vitis was found adhering to clothes and hands of workers carrying out customary cultural practices in the vineyard. Other arthropods, including Typhlodromus pyri, the main predator of C.vitis, were also passively transported by wind, rain and human activity.     

Infection of Tomicus piniperda (Col., Scolytidae) with Canningia tomici sp. n. (Microsporidia, Unikaryonidae)

Canningia tomici sp. n. (Microsporidia, Unikaryonidae) infects the midgut epithelium, the gut muscules, Malpighian tubules, connective tissues, adipose tissues and the gonads of the pine shoot beetle, Tomicus piniperda (L.) (Coleoptera, Scolytidae). The infection is present in populations of Tomicus piniperda in Europe and in the United States. Uninucleate oval single spores occur in two sizes: 2.8±0.4× 1.4±0.4m and 3.8±0.3×2.0±0.2m. The polar filament of this microsporidium is fixed subapically in a flat anchoring disc. The thick posterior lamellae of the binary polaroplast are asymmetric due to the lateral fixation of the polar filament.     

Induced Resistance to Cyst and Root-knot Nematodes in Cereals by DL-β-amino-n-butyric Acid

Foliar sprays and soil drenches with DL--amino-n-butyric acid (BABA) reduced the number of Heterodera avenae and H. latipons cysts on wheat and barley. Foliar sprays of wheat with 8000mgl^–1 BABA reduced the number of H. avenae cysts by 90%, whereas 2000mgl^–1 BABA was enough to reduce the number of H. latipons cysts by 79%. Multiple spray treatments with 2000mgl^–1 BABA at 10-day intervals reduced the number of H. avenae cysts on wheat and barley. A soil drench of wheat with 125mgl^–1 BABA reduced the number of H. latipons cysts by 93% and H. avenae cysts by 43%. Second-stage juveniles of these nematodes penetrated and formed syncytia in wheat roots soil-drenched with BABA. More adult males of H. avenae were produced in BABA (<250mg1^–1)-treated wheat roots (~76%) than in untreated roots (27%). Soil drenches with higher concentrations of BABA inhibited development of adult males and females. Several chemical elicitors of induced resistance were tested for their ability to reduce the number of H. avenae cysts on wheat. Only BABA was found to be an effective resistance inducer. The number of egg masses of an unidentified Meloidogyne sp. root-knot nematode, which infects only monocots, was also reduced by 95% by a soil drench of wheat with 500mgl^–1 BABA. Development of this nematode inside the BABA-treated roots was also inhibited.     

Shoot meristem tissue of tobacco inoculated with Cucumber mosaic virus is infected with the virus and subsequently recovers from infection by RNA silencing

Distribution of Cucumber mosaic virus (CMV) in shoot meristem tissue of CMV-inoculated tobacco was successively analyzed with immunohistochemical microscopy and in situ hybridization. CMV signals were detected in the tissue at 7 days postinoculation (dpi), but then they decreased and disappeared after 14dpi. Detailed observation confirmed CMV invasion of shoot apical meristem at 6–8dpi. Short interfering RNA corresponding to CMV RNAs was first detected at 7dpi and was detected up to 24dpi. These results suggest that the shoot meristem tissue is infected with CMV but subsequently recovers from the infection by RNA silencing.     

Winter mortality in sub-corticolous populations of Ips typographus (Coleoptera, Scolytidae) and its parasitoids in the south-eastern Alps

A study concerning the winter mortality of a sub-corticolous population of Ips typographus and its parasitoids has been carried out in the south-eastern Alps (Italy) during the winter 1997/98. Three attacked spruce trees were sampled three times (November, February and April) by collecting infested bark disks (1dm² each). All insects that emerged from the bark or died in the samples before emergence were counted. The mean number of living Ips typographus occurring under the bark decreases by 49% from November to April. Winter mortality mainly affects larval stages and young adults. The same trend was observed for the parasitoids Coeloides bostrychorum (48.5%) and Roptrocerus xylophagorum (47.5%).     

Infection of Tomicus piniperda (Col., Scolytidae) with Canningia tomici sp. n. (Microsporidia, Unikaryonidae)

Canningia tomici sp. n. (Microsporidia, Unikaryonidae) infects the midgut epithelium, the gut muscules, Malpighian tubules, connective tissues, adipose tissues and the gonads of the pine shoot beetle, Tomicus piniperda (L.) (Coleoptera, Scolytidae). The infection is present in populations of Tomicus piniperda in Europe and in the United States. Uninucleate oval single spores occur in two sizes: 2.8±0.4× 1.4±0.4m and 3.8±0.3×2.0±0.2m. The polar filament of this microsporidium is fixed subapically in a flat anchoring disc. The thick posterior lamellae of the binary polaroplast are asymmetric due to the lateral fixation of the polar filament.     

Efficiency of the mixed biocide GCSC-BtA against vegetable pests of different arthropod orders in the south-eastern China

The present paper deals with the efficiency of the mixed biocide GCSC-BtA against vegetable pests of five orders of arthropod insects in the south-eastern China, i.e., diamondback moth, Plutella xylostella (L.); green peach aphid, Myzus persicae (Sulzer); yellow-fanded flea beetle, Phyllotreta vittata Fabricius; vegetable leaf miner, Phytomyza nigricornis Macquart; and vegetable spider mite, Tetranychus neocaledonicus Andre. The results show that the mixed biocide GCSC-BtA has a higher efficacy of control against all of the five pests than the commercial biocide B.t. and insecticide Diazinon. GCSC-BtA gave a higher mortality value of 92.18% against P. xylostella as compared to the biocide B.t. with a value of 64.70% and Diazinon with a value of 50.00%. Similar mortalities were obtained with the other four pests regarding the efficacy of the three products tested against them.The concentration-mortality regression equations were calculated by the probit analysis, from which the average mortalities were calculated. The values calculated for GCSC-BtA were 81.13% for P. xylostella, 75% for M. persicae and T. neocaledonicus, 69.23% for P. vittata, and 61.03% for P. nigricornis as compared to those caused by Diazinon: 33.33%, 27.42%, 66.31%, 44.54%, 19.65% for P. xylostella, M. persicae, P. vittata, P. nigricornis, and T. neocaledonicus, respectively. Based on LC₅₀, P. xylostella was most susceptible to GCSC-BtA with a LC₅₀ value of 0.3445, followed by B.t. with a value of 0.8404 and Diazinon with a value of 1.3160. The LC₅₀ value of GCSC-BtA for M. persicae was 0.4660 which was about three times less than that of Diazinon. LC₅₀ value of GCSC-BtA for P. vittata was 0.3954 which was about the same as that of Diazinon. P. nigricornis and T. neocaledonicus were also much more susceptible to GCSC-BtA than to B.t. and Diazinon. It is concluded that GCSC-BtA kills more target pests than B.t. and is more effective than Diazinon in controlling the vegetable pests.     

Studies on insects and mites causing galls on the leaves of purple willow Salix purpurea L. in the Ojców National Park in Poland

Analytical studies were conducted on the species composition of galls occurring on the leaves of Salix purpurea L. in the Ojców National Park in 1999–2000. The species composition of galls was identical in two consecutive years. Galls were caused by 5species (a total of 480galls). The most numerous species in both years was Pontania viminalis (L.). Index of occurrence frequency and Agrell's index of species co-existence have been calculated. This index reached the highest values in case of Pontania dolichura (Thoms.) and P. viminalis (L.) in 1999 as well as Phyllocolpa leucaspis (Tischb.) and P. viminalis (L.) in 2000.     

Investigations on side-effects of the mixed biocide GCSC-BtA on different predators of Plutella xylostella (L.) (Lep., Plutellidae) in southeastern China

The present paper deals with laboratory studies on side-effects of the mixed biocide GCSC-BtA on Plutella xylostella (L.) (Lep., Plutellidae) and its predators, e.g. Amblyseius longispinosus (Evans) (Acari, Phytoseiidae), Erigonidium graminicola (Sundvall) (Araneae, Linyphiidae), Orius similis Zheng (Het., Anthocoridae) and Coccinella septempunctata L.(Col., Coccinellidae), in comparison to the commercial insecticides, e.g. Abamectin, Tebufenozide, Dichlorvos, Cartap and Lambda-cyhalothrin.The results showed that GCSC-BtA was highly toxic to the 3^rd instar of P. xylostella with 91.18% mortality, followed by Cartap with 84.38%, Abamectin with 78.00%, Tebufenozide with 75.57%, Lambda-cyhalothrin with 63.75% and Dichlorvos with 50.86% mortality. On the other hand, GCSC-BtA was found to be comparatively less toxic to the predators, causing 31.11%, 13.33%, 11.54% and 6.00% mortalities in A. longispinosus, E. graminicola, O. similis and C. septempunctata, respectively. For comparison, the mortalities recorded for Abamectin, Tebufenozide, Dichlorvos, Cartap, Lambda-cyhalothrin were 72.94%, 55.55%, 70.00%, 53.26% and 98.85% in A. longispinosus, 46.51%, 55.10%, 60.00%, 46.00% and 73.68% in E. graminicola, 22.00%, 16.00%, 35.71%, 26.78% and 81.03% in O. similis, 15.55%, 19.64%, 28.00%, 16.66% and 41.79% in C. septempunctata, respectively.     

Detection of Xanthomonas sp., the Causal Agent of Onion Bacterial Blight, in Onion Seeds Using a Newly Developed Semi-selective Isolation Medium

Onion bacterial blight, caused by Xanthomonas sp., is a potentially severe disease in several tropical and subtropical areas. Although little research has been undertaken on this pathosystem, seed transmission of the pathogen has been hypothesized. Because of an important bacterial microflora naturally associated with onion seeds, detection of the pathogen is difficult using non-selective agar media. A new semi-selective medium, whose selectivity was obtained by a combination of four antibiotics, was developed. The new NCTM1 medium contained (per liter) yeast extract 7g, peptone 7g, glucose 7g, agar 15g, neomycin 10mg, cephalexin 30mg, trimethoprime 3mg, pivmecillinam 100mg and propiconazole 20mg. Plating efficiencies, using 16 pure cultures of the pathogen, ranged from 79% to 142%, with an average of 110% compared to the basal medium. All onion Xanthomonas sp. strains from several countries grew on NCTM1 medium. The pathogen was repeatedly isolated using this medium from seed samples containing approximately 10⁶ saprophytic bacteria per gram, as well as from symptomless plant material. Xanthomonas sp. was detected only in seeds originating from one infected seed production site. This is the first report of selective isolation of Xanthomonas sp. from onion seeds. NCTM1 medium should be a valuable tool to study the ecology and epidemiology of Xanthomonas sp. causing onion bacterial blight.