Woody Gall symptoms in different citrus species and varieties artificially colonized by the citrus brown aphid Toxoptera citricidus

Seedlings of 41 different citrus species and varieties were massively colonized with the citrus brown aphid Toxoptera citricidus, obtained from Pêra sweet orange (Citrus sinensis) trees, presenting symptoms of the "Capão Bonito" complex of the Citrus tristeza virus (CTV). The objective was to evaluate resistance or tolerance of the varieties to that virus complex, but even after eight months of inoculation no stem pitting was observed in the plants. Otherwise, the presence of galls similar to those induced by the vein enation-woody gall disease was observed in 73% of the plants of Volkamer Palermo (Citrus volkameriana), 60% of the Volkamer Catania 2, 2% of the Rangpur Lime D.22.30 (Citrus limonia), 13% of the Volkamer Australian Red, the Rangpur Lime hybrid, the Orlando tangelo (Citrus reticulata x Citrus paradisi) and the Florida Rough lemon (C. jambhiri), and 7% of the Carrizo citrange (Poncirus trifoliata x Citrus sinensis). The highest incidence and the largest gall size were observed in the Palermo Volkamer showing that this clone would be the most suitable to be used as an indicator plant in biological indexing tests for the disease. There are no previous reports in the literature about the occurrence of woody galls in Orlando tangelo and Carrizo citrange.

Identification of Phaeoisariopsis griseola pathotypes from five States in Brazil

Due to the increased importance of angular leaf spot of common bean (Phaseolus vulgaris) in Brazil, monitoring the pathogenic variability of its causal agent (Phaeoisariopsis griseola) is the best strategy for a breeding program aimed at developing resistant genotypes. Fifty one isolates of P. griseola collected in five Brazilian States were tested on a set of 12 international differential cultivars in the greenhouse. When inoculated plants showed symptoms but no sporulation was observed, they were transferred to a moist chamber for approximately 20-24 h. After this period of time, if no sporulation was observed, the plants were considered resistant; otherwise, they were considered susceptible. From the fifty-one tested isolates, seven different pathotypes were identified. No Andean pathotypes were identified; consequently, all isolates were classified as Middle American pathotypes. Pathotype 63-31 was the most widespread. Pathotype 63-63 overcame resistance genes present in all differential cultivars and also the resistance gene(s) present in the cultivar AND 277. This fact has important implications for breeding angular leaf spot resistance in beans, and suggests that searching for new resistance genes to angular leaf spot must be pursued.

Use of semi-seletive media for detection of Sclerotinia sclerotiorum on bean and soybean seeds

This work was aimed at evaluating the possibility of using bromophenol blue as an indicator for detecting the presence of Sclerotinia sclerotiorum in the seeds of dry-beans (Phaseolus vulgaris) and soybean (Glycine max), through incubation of the seeds on an agar medium and "blotter" substrates. The seeds were artificially inoculated with four S. sclerotiorum isolates, plated on the agar medium, named Neon, and on modified Neon agar media all incubated at 14 and 20 ºC for seven days in the dark. Half of the seeds inoculated were surface desinfested prior to plating on the medium. The seeds showing change of colour in the medium, from blue to light yellow, as well as formation of typical mycelium and sclerotia in some cases, were considered to be infected or contaminated by S. sclerotiorum. The two incubation temperatures compared did not show significant (P<0.05) differences in detection level for most of the isolates tested on the different media. According to results obtained in this study, the Neon agar medium with incubation at 14 or 20 ºC has proved to be a reliable and quick method for the detection of S. sclerotiorum mycelium in naturally infected seeds of bean and soybean.

Telomere and microsatellite primers reveal diversity among Sclerotinia sclerotiorum isolates from Brazil

Sclerotinia sclerotiorum, the causal agent of white mold, is a problem of winter bean (Phaseolus vulgaris) production in Brazil under center-pivot irrigation. Isolates of S. sclerotiorum were obtained from a center-pivot-irrigated field near Guaíra-SP, Brazil. Mycelial compatibility group (MCG) studies revealed the presence of only two MCG. PCR/RFLP analysis of the ITS1-5.8S-ITS2 ribosomal subunit regions of these field isolates of S. sclerotiorum failed to show any genetic differences between these two MCGs. DNA amplification with a chromosomal telomere sequence-based primer and one microsatellite primer revealed genetic polymorphisms among isolates within the same MCG. Isolates taken from beans and two other crops from another region of Brazil showed the same two MCG and had identical banding patterns for the telomere and microsatellite primers. These findings support the use of telomere sequence-based primers for revealing genotypic differences among S. sclerotiorum isolates.

Effects of single and double infections with Potato virus X and Tobacco mosaic virus on disease development, plant growth, and virus accumulation in tomato

The tomato cv. Fukuju nº. 2 was used for studying the effect of single and double infections with Potato virus X (PVX) and Tobacco mosaic virus (TMV). Mixed infection resulted in a synergistic increase of disease severity, where more growth reduction was seen with simultaneous inoculations than with sequential inoculations at four-day intervals. At five and 12 days post-inoculation, the increased severity of the disease coincided with enhancement of virus accumulation in the rapidly expanding upper leaves. The PVX concentration in leaves nº 5 to 7 of doubly infected plants was three to six fold that of singly infected ones, as determined by DAS-ELISA. Mixed infection with the L strain led to higher enhancement of PVX than with the TMV-L11A strain. The concentration of TMV-L was lower in double infection and significantly higher than TMV-L11A in both singly and doubly infected plants. Analyses of the PVX ORF2 by Western blot and Northern hybridization revealed the pattern of accumulation of the 25 kDa protein and the RNAs, respectively, following those of the virion and coat protein. The strain TMV-L11A overcame the resistance gene in cv. GCR 237 (Tm-1). In the upper leaf nº. 8, the concentration of PVX was three times higher in plants with mixed infection than with L11A. The concentrations of the L and OM (TMV strains) in both singly and doubly infected plants were at very low levels, and the synergistic effect on PVX concentration and disease severity was not observed.

Genetic analyses of Rhizoctonia solani isolates from Phaseolus vulgaris grown in the Atlantic Rainforest Region of São Paulo, Brazil

Rhizoctonia solani isolates obtained from common beans (Phaseolus vulgaris) grown in the mountainous Atlantic Rainforest (Mata Atlântica) region of São Paulo, Brazil, were analyzed to determine their genetic diversity using internal transcribed spacer (ITS), microsatellite and telomere sequence-based PCR primers. Restriction digestion of the ITS1/5.8S/ITS2 ribosomal regions yielded unique banding patterns specific for AG4 and its subgroups. The ITS restriction digestion (ITS/RFLP), telomere and microsatellite primers identified five to 11 genotypes within the isolates of R. solani. While all isolates were pathogenic on beans, there was no correlation found between genotypic differences and pathogenicity. The different PCR primers revealed a number of isolates that were genetically similar. Some of these genetic groups were supported by more than one of the primers utilized in this study, thus confirming their relationship.

Effect of population densities of Heterodera glycines race 3 on leaf area, photosynthesis and yield of soybean

The effect of Heterodera glycines on photosynthesis, leaf area and yield of soybean (Glycine max) was studied in two experiments carried out under greenhouse condition. Soybean seeds were sown in 1.5 l (Experiment 1) or 5.0 l (Experiment 2) clay pots filled with a mixture of field soil + sand (1:1) sterilized with methyl bromide. Eight days after sowing, seedlings were thinned to one per pot, and one day later inoculated with 0; 1.200; 3.600; 10.800; 32.400 or 97.200 J2 juveniles of H. glycines. Experiment 1 was carried out during the first 45 days of the inoculation while Experiment 2 was conducted during the whole cycle of the crop. Measurements of photosynthetic rate, stomatic conductance, chlorophyll fluorescence, leaf color, leaf area, and chlorophyll leaf content were taken at ten-day intervals throughout the experiments. Data on fresh root weight, top dry weight, grain yield, number of eggs/gram of roots, and nematode reproduction factor were obtained at the end of the trials. Each treatment was replicated ten times. There was a marked reduction in both photosynthetic rate and chlorophyll content, as well as an evident yellowing of the leaves of the infected plants. Even at the lowest Pi, the effects of H. glycines on the top dry weight or grain yield were quite severe. Despite the parasitism, soybean yield was highly correlated with the integrated leaf area and, accordingly, the use of this parameter was suggested for the design of potential damage prediction models that include physiological aspects of nematode-diseased plants.

Expression of resistance in rice hybrids to Pyricularia grisea

Thirty-nine rice (Oryza sativa) hybrids and their restorers were assessed for vertical resistance to Pyricularia grisea in the rice blast nursery, and in artificial inoculation tests with two pathotypes, under controlled greenhouse conditions. The hybrids were developed from cytoplasmic genetic male sterile lines 046I and IR 58025A, derived from WA cytoplasm. In the rice blast nursery all hybrids showed susceptible reaction varying from 5 to 9. Compatible and incompatible leaf blast reactions of hybrids to two pathotypes, IC-1 and IB-45, were observed in inoculation tests. A majority of the hybrids were resistant when the restorer was resistant. However, seven of the 25 F1 hybrids exhibited susceptible reactions even when one of the parents was resistant to a pathotype. The partial resistance of 11 hybrids and their parents that showed compatible reactions to two pathotypes was analyzed. Differential interaction between isolates and genotypes was observed for partial resistance in relation to both disease severity and lesion number indicating the specific nature of partial resistance.

Podridão basal pós-colheita do coco verde no estado do Ceará

A new post-harvest disease of coconut (Cocos nucifera) was recently detected in fruits exported to European countries. The main symptoms are the blacking and cracking of basal parts of the fruits. Water oozing may occurs as the infection progresses. The fungus Lasiodiplodia theobromae has been frequently isolated from the lesions. Inoculation tests proved that this pathogen is the causal agent of the coconut fruit basal rot. This is the first occurrence of this post-harvest disease in Brazil.

Genetic and phenotypic characterization of isolates of Pyricularia grisea from the rice cultivars epagri 108 and 109 in the State of Tocantins

An epidemic of rice (Oryza sativa) blast occurred on cultivars Epagri 108 and 109 in the municipalities of Lagoa da Confusão and Duerê in the State of Tocantins, during the rice-growing season 1998-99. DNA fingerprinting and virulence phenotype analysis were utilized to determine the diversity of Pyricularia grisea isolates collected from these cultivars in one epidemic year. Rep-PCR analysis of isolates was done by using two primer sequences from Pot2. Two distinct fingerprint groups or lineages were identified among 53 isolates collected from nine different commercial fields. The virulence pattern of isolates retrieved from these two cultivars was analyzed in artificial inoculation tests utilizing 32 genotypes in the greenhouse. A dendrogram constructed from virulence phenotype data showed a single group considering 77% similarity level. The predominant pathotype IB-45 was represented by 47 of the 53 isolates corresponding to 83%. Four other pathotypes (IB-1, IB-9, IB-13 and IB-41) were identified at random among the isolates from these cultivars. There was no relation between rep-PCR grouping and pathotypes. The results showed that the isolates of P. grisea recovered from cultivars Epagri108 and 109 in farmers' fields had narrow phenotypic and genetic diversity. The blast outbreak on these two cultivars one year after their introduction could be attributed to the new pathotype IB-45 or its increase, which was hitherto existing in low frequency.

Ocorrência de Bipolaris maydis causando mancha foliar em Paspalum atratum cv. Pojuca no Brasil

Bipolaris maydis was consistently isolated from infected Paspalum atratum cv. Pojuca plants showing leaf spot symptoms in the Cerrado of Brazil, in 2002. Pathogenicity tests under greenhouse conditions and subsequent reisolations of B. maydis from artificially inoculated Pojuca seedlings confirmed the hypothesis that this fungus was the causal agent of the disease. Symptoms of leaf spot appeared four days after inoculation in 100% of the inoculated Pojuca plants. All seven species of grasses evaluated were susceptible to B. maydis. The occurrence of leaf spot of Pojuca caused by B. maydis is reported for the first time in Brazil.

Essential oil of mustard to control Rhizoctonia solani causing seedling damping off and seedling blight in nursery

The essential oil extracted from mustard (Brassica rapa) seeds was evaluated for its effect on suppression of Rhizoctonia solani growth in vitro, and in field soils, for reducing saprophytic substrate colonization and seedling damping off and blight using snap beans as indicator plant, the in vitro growth was completely inhibited at a concentration of 50 mul/l. The saprophytic substrate colonization in soils 24 h after treatment was drastically reduced to 45% at 150 mul/kg soil concentration, in contrast to 100% colonization at concentrations of 0, 50, or 75 mul/kg. This recovery rate gradually declined to 6% and 60%, respectively, in nine days. A control of pre and post-emergence seedling damping off and blight in common beans (Phaseolus vulgaris), without any apparent phytotoxic effect was achieved by irrigating R. solani infested soils with water containing the emulsified essential oil to provide 150 mul/l soil volume ten days prior to planting, gave over 95%. The effect of the mustard essential oil was not influenced by the physical soil texture, and it appears to be a good substitute for methyl bromide fumigation in nurseries for seedling production.

Transgenic passionfruit expressing RNA derived from Cowpea aphid-borne mosaic virus is resistant to passionfruit woodiness disease

Sixteen transgenic yellow passionfruit (Passiflora spp.) plants (R0) were obtained which express a non-translatable transgenic RNA corresponding to the 3' region of the NIb gene and the 5' region of the CP gene, derived from the genome of a Brazilian isolate of Cowpea aphid-borne mosaic virus (CABMV). The transgenic plants were propagated by stem cuttings and challenged by sap inoculation with isolates CABMV-MG1 and CABMV-PE1. One transgenic plant (TE5-10) was resistant to the isolate CABMV-MG1, but susceptible to CABMV-PE1. The remaining transgenic plants developed systemic symptoms, equal to non-transformed plants, when inoculated with either isolate. The absence of virus in TE5-10 plants was confirmed by indirect ELISA. Transcription analysis of the transgene demonstrated that the TE5-10 plant did not accumulate transgenic mRNA, even before inoculation. After inoculation, viral RNA was only detected in plants inoculated with CABMV-PE1. These results confirm that the transgenic plant TE5-10 is resistant to isolate CABMV-MG1, and suggest that the resistance mechanism is post-transcriptional gene silencing, which is already activated in the transgenic plants before virus inoculation.

AFLP markers differentiate isolates of Colletotrichum gossypii from C. gossypii var. cephalosporioides

Fungal diseases in cotton (Gossypium hirsutum), such as anthracnose caused by Colletotrichum gossypii and ramulose caused by C. gossypii var. cephalosporioides, are responsible for large yield losses. These pathogens are seed borne and morphologically similar although they induce different symptoms, which can lead to misdiagnosis using the blotter testing method. The present study was carried out to assess the viability of using Amplified Fragment Length Polymorphism (AFLP) markers to differentiate these pathogens. Five isolates, for each pathogen, were classified according to pathogenicity on cotton plants, and mycelial growth morphology. Conidial suspensions were sprayed on 30-day-old cotton plants and the symptoms assessed ten and 40 days after inoculation. For growth morphology 200 cottonseeds were inoculated with seven-day-old pure cultures, and the mycelial traits observed under a stereoscopic microscope seven days after inoculation. The DNA for AFLP analysis was obtained from seven-day-old fungal mycelia grown in liquid medium, using the Dneasy Qiagen protocol. Using the AFLP technique 318 polymorphic bands were selected to estimate similarities using Dice's Coefficient. The results clearly distinguished between ramulose and anthracnose isolates, which agreed with morphological and pathogenicity testing.

Influence of internal seedborne Fusarium semitectum on cotton seedlings

Fusarium semitectum was found to be the major seed colonizing fungus in the commercial acid delinted cotton (Gossypium hirsutum) seed lots. There was no correlation, however, between its incidence and seedling emergence and disease symptoms on the emerged seedlings in autoclaved sand. Inoculation technique simulating internally seedborne nature of the fungus showed that the observed non-correlation might be related to the threshold level of seed coat colonization. The internally seedborne inoculum besides reducing seedling emergence, incited an array of symptoms on the emerged seedlings, which ranged from negative geotropism, leaf tearing, collar rot leading to dry root rot and seedling mortality. The dry root rot continued to develop on the plants surviving the seedling phase. The collar rot symptoms can be confused with those caused by Rhizoctonia solani.