Potential for Biocontrol of Monosporascus Root Rot/vine Decline Under Greenhouse Conditions using Hypovirulent Isolates of Monosporascus cannonballus

Monosporascus root rot/vine decline (MRR/VD) causes root necrosis and severe stunting of muskmelon and watermelon plants in several countries around the world. MRR/VD is caused by the soilborne ascomycete fungus, Monosporascus cannonballus. Currently, there are few options available for control of MRR/VD. This research describes experiments to test the possibility of using naturally occurring M. cannonballus isolates containing double-stranded RNA (dsRNA) for the biological control of MRR/VD. These isolates often develop a degenerate phenotype characterized by slow growth and reduced ascospore production. In addition, these degenerate isolates are hypovirulent on muskmelon. Plants co-inoculated with a hypovirulent, dsRNA⁺ isolate (Tx93-449⁺) and a virulent, dsRNA^- isolate (Az90-33^-) at an inoculum ratio of 10:1 (hypovirulent:virulent) were indistinguishable from the uninoculated plants in greenhouse pathogenicity trials. In vitro infection assays using fluorescence microscopy on aniline-stained muskmelon roots suggested that although the hypovirulent dsRNA⁺ isolate Tx93-449⁺ penetrated and partially colonized roots of the seedlings, it was not as efficient in colonizing the roots as the virulent, dsRNA^- isolate Az90-33^-. While more extensive experiments are needed, these data suggest that hypovirulent dsRNA⁺ isolates of M. cannonballus have potential for development as biological control agents to reduce disease pressure associated with MRR/VD.     

Effect of Fungicides on Fusarium Head Blight and Deoxynivalenol Content in Durum Wheat Grain

In 1998–99 and 1999–2000 six trials were conducted to evaluate the effect of fungicides on Fusarium head blight in the field, on infected kernels and deoxynivalenol (DON) concentration in grain. A single application of prochloraz, tebuconazole, epoxiconazole or bromuconazole, applied to durum wheat varieties at the manufacturer's recommended dose at the beginning of anthesis stage, provided good control of the disease when infective pressure in the field was low to medium, and when the main pathogens were F. graminearum and F. culmorum. Kresoxim-methyl showed a low efficacy at controlling the disease. Tebuconazole, prochloraz and bromuconazole were effective at controlling F. graminearum and F. culmorum, while kresoxim-methyl was not effective in reducing Fusarium infected kernels. DON concentration in grain of cultivars inoculated with F. graminearum and F. culmorum was high, averaging 4.2mgkg^–1 (untreated control). Tebuconazole, prochloraz and bromuconazole reduced DON concentration by 43%, while epoxiconazole was ineffective. DON concentration in kernels of naturally infected cultivars was 1.95mgkg^–1, a concentration which exceeds the 1mgkg^–1 maximum level of contamination allowed in the United States. Furthermore prochloraz, bromuconazole and tebuconazole applications, in the naturally inoculated trials, reduced DON concentration from 73% to 96%, while epoxiconazole showed the lowest effectiveness. Moreover, a positive linear correlation between Fusarium infected grains and the DON concentration was observed.     

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.     

Pathogenic variation in poplar rust <Emphasis Type="Italic">Melampsora larici-populina</Emphasis> from England

Using a leaf disc method, 19 isolates of the poplar rust, Melampsora larici-populina , and one isolate of M.populnea from England were inoculated on to 25 poplar clones belonging to Populus nigra and P.trichocarpa, and hybrids between P. deltoides and P. nigra, P. deltoidesand P. trichocarpa, P.tacamahaca and P.trichocarpa, and P. alba and P. tremula. Disease was scored based on the pustule area and inoculum density. In terms of whether sporulating uredinia formed, the 19 isolates showed seven different patterns to the tested poplar clones. The majority of the rust isolates infected P. nigra P3090 and Vereecken, P.nigra×P. deltoides Casale and Tasman, P. tacamahaca×trichocarpa 36 and Balsam Spire, and P.trichocarpa Blom. Populus trichocarpa×P. deltoides 69039/4 was infected by only three isolates collected from southern England. No visible symptoms appeared on P. alba ×P. tremulaTower and P.trichcarpa×P. deltoides×P. deltoides76028/5 in inoculations with M. larici-populina isolates. Populus alba×P.tremula Tower was infected only by M. populnea. When M. larici-populina isolates were tested using AFLP, no differences were found either between isolates from different geographical regions or between those having narrow spectrum of virulence and those showing wide spectrum of virulence on the tested clones. The results suggest that the UK rust populations possess virulences which were found in races E1, E2, E3 and E4 in continental Europe and that rust having virulence patterns similar to race E4 has occurred in UK poplar plantations since 1996.     

The Effect of Soil Moisture and Cabbage Amendment on the Thermoinactivation of Phytophthora nicotianae

The analysis of the effect of soil water matric potential and temperature regimes on the inactivation of chlamydospores of Phytophthora nicotianae in cabbage amended soils was evaluated using three matric potentials (0, -10, and -30kPa), temperature regimes of 1.5h at 44°C, 5h at 41°C and 8h at 35°C, or 3h at 47°C, 5h at 44°C and 8h at 35°C, with a baseline temperature of 25°C during the rest of the day. The results indicated that survival of P. nicotianae was lowest in saturated soil; and as temperature increased, survival of the pathogen decreased at all soil water matric potentials evaluated. Cabbage amendments can enhance the effect of the heat treatment, further decreasing the pathogen population. The soil water matric potentials evaluated represent optimum levels for the study of thermal inactivation. However, under field conditions lower potentials may be found. Extending the range of soil water matric potentials and the treatment time would allow better comparisons with the field data. There is a clear indication that one irrigation period prior to solarization would provide enough moisture to inactivate the primary inoculum of P. nicotianae in the top soil under field conditions; however, other factors may affect the effectiveness of solarization, reducing or enhancing its potential.     

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.     

Environmental factors influencing sporocarp formation in <Emphasis Type="Italic">Typhula ishikariensis</Emphasis>

Environmental factors influencing sporocarp formation in Typhula ishikariensis were studied under controlled conditions. Sporocarp formation in T. ishikariensis was divided into two stages: stipe elongation from the sclerotium and fertile head development at the tip of the stipe. Factors required for each stage differed. At the stipe elongation stage, low temperature (10°/5°C; day/night) and high humidity were important, but light was not required. In contrast, at the fertile head stage, light and moderate day length (8h/day) were essential. Fertile heads developed at 46µEm^–2s^–1; and high intensity (137µEm^–2s^–1) did not suppress development. Moreover, adding unsterilized soil to the sea sand medium accelerated sporocarp formation. These findings imply that the sclerotium of T. ishikariensis recognizes several physical factors for sporocarp formation. Sporocarps of T. ishikariensis developed within 4 weeks after incubation under optimal conditions. The sporocarp produced basidiospores, and differential mating incompatibility was confirmed among monokaryons derived from basidiospores produced under artificial conditions. This method should be useful for obtaining monokaryons for genetic studies of T. ishikariensis.     

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.     

Interactions between Plasmopara helianthi, Glomus mosseae and Two Plant Activators in Sunflower Plants

Interactions between Plasmopara helianthi, Glomus mosseae and two plant activators DL--amino-n-butyric acid (BABA) and CGA 245704 (acibenzolar-S-methyl (BTH)) in sunflower plants susceptible to downy mildew were studied in four experiments using different methods of treatment and pathogen inoculation. Both chemicals were applied as soil drenches and foliar sprays, whereas P. helianthi infection was obtained by root and cotyledon inoculations of the seedlings. Soil drenches at the rates of 50 and 100mgkg^–1 soil of BABA and BTH given 1 and 3 days before P. helianthi inoculation, respectively to mycorrhizal plants, provided moderate protection against the pathogen (about 50–55%). Morphological changes and decrease in mycorrhizal colonization in roots of BTH-treated plants and in BTH-treated mycorrhizal plants were also observed. Delay in the emergence and reduction of the root systems were more evident at the highest concentration but decreased with time. These effects were absent with the BABA treatment.Foliar spray treatment of BABA and BTH, applied at 4000 and 200µgml^–1, respectively (1 day post-inoculation) to mycorrhizal plants provided good protection (about 80%) against P. helianthi foliar infections. No effects on mycorrhizal colonization or on root systems were observed. In vitro tests on the effect of the compounds on the mycorrhizal fungus showed that the germination of G. mosseae sporocarps increased with BABA treatment whereas it was greatly inhibited by BTH treatment.     

Effects of light conditions on prodigiosin stability in the biocontrol bacterium Serratia marcescens strain B2

Serratia marcescens strain B2 is a potential biocontrol agent that produces the antibiotic pigment prodigiosin. When this strain was incubated under white or blue light conditions (<100µmol m^–2s^–1), prodigiosin concentration in bacterial cells decreased, but growth did not. However, red and far-red light had no effect on prodigiosin concentration in bacterial cells. Purified prodigiosin was degraded under white or blue light conditions but was not degraded under red and far-red light. Because white and blue light appeared to affect the stability of prodigiosin itself, light conditions may affect the suppressive effects of the biocontrol agent S. marcescens strain B2.     

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.     

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.     

Role of Salicylic Acid in Systemic Resistance Induced by Pseudomonas spp. Against Pythium aphanidermatum in Cucumber Roots

Pseudomonas corrugata strain 13 and P. aureofaciens strain 63-28, applied to roots, induced systemic resistance against Pythium aphanidermatum in cucumber roots. Salicylic acid (SA) from bacterial culture or plant tissues was quantified by high performance liquid chromatography. Both strains produced SA in King's B broth and also induced cucumber root to accumulate endogenous SA one day after bacterial inoculation. Using a split root system, more SA accumulated in roots treated with bacteria than in distant roots on the opposite side of the root system in the first two days, but this difference disappeared after 3–4 days. SA levels were significantly higher in plants treated with bacteria compared to the split control, from one to five days after bacterization. SA did not inhibit mycelial growth of Pythium aphanidermatum at 100–200µgml^–1 in vitro, but higher levels inhibited mycelial growth. Zoospore germination increased at concentrations of 10–500µgml^–1, but decreased at 1000µgml^–1 compared to lower concentrations. Exogenously applied SA failed to induce local or systemic resistance against a challenge infection by the pathogen in planta. The results of this study show that exogenous applied SA does not induce systemic resistance to cucumber root rot caused by P. aphanidermatum, but endogenous SA accumulation in cucumber roots may be involved in induced systemic resistance.     

Three distinct groups of isolates of <Emphasis Type="Italic">Tomato yellow leaf curl virus</Emphasis> in Japan and construction of an infectious clone

Complete nucleotide sequences of eight Japanese isolates of Tomato yellow leaf curl virus (TYLCV) were determined and compared with four TYLCV isolates already reported. These isolates separated into three groups – Shizuoka (Sz), Aichi (Ai), Nagasaki (Ng) – and had 99% identities within the groups. Full-length molecules of DNA-A of group Sz consist of 2791nt and those of group Ai contain 2787nt. Both were closely related to TYLCV-Is.M, although those of group Ng had 2793nt and were more closely related to TYLCV-Is. Comparison of common sequences of isolates belonging to groups Sz and Ai had substitutions of 4nt in the intergenic region and nonsynonymous substitutions at open reading frames between the groups. None of the isolates tested had DNA molecules. Agroinfection of four plant species with a DNA-A dimeric infectious clone of TYLCV-SzY, a member of group Sz, resulted in systemic infection. Tomato plants then developed typical yellow leaf curl symptoms.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB116629, AB116630, AB116631, AB116632, AB116633, AB116634, AB116635, and AB116636     

Interactions between the striped stem borer Chilo suppressalis (Walk.) (Lep., Pyralidae) larvae and rice plants in response to nitrogen fertilization

A screenhouse experiment was conducted to examine the damage and compensation in rice plants when injured by the striped stem borer, Chilo suppressalis (Walker), larvae at tillering stage, as well as larval survival and development of the insect at different nitrogen (N) fertilization levels. Potted plants were fertilized at late seedling stage at the rates 0, 200, 400, 600 and 800mgN/pot, respectively. More deadheads were caused as fertilization increased. Plants compensated well for injury at the fertilization concentrations of 200 and 400mgN/pot by producing new tillers, but such compensation did not take place at 600 and 800mgN/pot. Two weeks after infestation, the highest number of remaining healthy tillers was found in plants fertilized at 400mgN/pot. Larval survival varied little among the treatments 200 to 800mgN/pot. Larval weight attainment and/or developmental rate increased with increasing fertilization level from 200 to 600mgN/pot, but both declined rapidly as fertilization reached 800mgN/pot, indicating the great dependence of plant suitability on N fertilization levels. Conclusively, both the compensation response of rice plants and their suitability for C. suppressalis larvae could be significantly affected by N fertilization levels.     

Interactions between the striped stem borer Chilo suppressalis (Walk.) (Lep., Pyralidae) larvae and rice plants in response to nitrogen fertilization

A screenhouse experiment was conducted to examine the damage and compensation in rice plants when injured by the striped stem borer, Chilo suppressalis (Walker), larvae at tillering stage, as well as larval survival and development of the insect at different nitrogen (N) fertilization levels. Potted plants were fertilized at late seedling stage at the rates 0, 200, 400, 600 and 800mgN/pot, respectively. More deadheads were caused as fertilization increased. Plants compensated well for injury at the fertilization concentrations of 200 and 400mgN/pot by producing new tillers, but such compensation did not take place at 600 and 800mgN/pot. Two weeks after infestation, the highest number of remaining healthy tillers was found in plants fertilized at 400mgN/pot. Larval survival varied little among the treatments 200 to 800mgN/pot. Larval weight attainment and/or developmental rate increased with increasing fertilization level from 200 to 600mgN/pot, but both declined rapidly as fertilization reached 800mgN/pot, indicating the great dependence of plant suitability on N fertilization levels. Conclusively, both the compensation response of rice plants and their suitability for C. suppressalis larvae could be significantly affected by N fertilization levels.     

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.     

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.     

Effects of Host and Microbial Factors on Development of Clonostachys rosea and Control of Botrytis cinerea in Rose

Development of Clonostachys rosea in rose leaves and petals and control of Botrytis cinerea by the agent were investigated. C. rosea germinated, established endophytic growth, and sporulated abundantly whether the tissues were mature, senescent or dead when inoculated. Germination incidence was moderate on mature and senescent leaves (47% and 35%) and petals (31% and 43%), and high (>98%) on dead tissues. Sporulation of C. rosea in tissues inoculated when mature, senescent or dead averaged 41%, 61%, and 75% in leaves, and 48%, 87% and 53% in petals. When leaves were wounded with needles before inoculation, germination of C. rosea increased from 45–56% to 90–92%, but sporulation became high (>75%) regardless of wounds. When leaves were inoculated with C. rosea at 0–24h after wounding and subsequently with B. cinerea, germination of the pathogen was reduced by 25–41% and sporulation by 99%. A humid period prior to inoculation of senescent or dead leaves promoted communities of indigenous fungi, reduced sporulation of C. rosea and B. cinerea, and, in dead leaves, increased control of the pathogen associated with C. rosea. Applied at high density, isolates of indigenous Penicillium sp. and Alternaria alternata from rose interacted with C. rosea and reduced control of the pathogen by 16% and 21%, respectively. In conclusion, C. rosea markedly suppressed sporulation of B. cinerea in rose leaves and petals regardless of developmental stage, minor wounds, and natural densities of microflora. This versatility should allow C. rosea to effectively control inoculum production of B. cinerea in rose production systems.     

PCR-based specific detection of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> race 4 strains

Two primer sets were designed based on the sequence of polymorphic bands that were derived from repetitive sequence-based polymerase chain reaction (rep-PCR) fingerprinting and specifically detected in Ralstonia solanacearum race 4 strains (ginger, mioga, and curcuma isolates). One primer set (AKIF-AKIR) amplified a single band (165bp) from genomic DNA obtained from all mioga and curcuma and some ginger isolates; another set (21F-21R) amplified one band (125bp) from the other ginger isolates. These primer sets did not amplify the bands from genomic DNA of other R. solanacearum strains or of other related bacteria. PCR detection limit for the pathogen was 2 × 10²cfu.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB118756 and AB118757