Full establishment of arbuscular mycorrhizal symbiosis in rice occurs independently of enzymatic jasmonate biosynthesis.

Development of the mutualistic arbuscular mycorrhiza (AM) symbiosis between most land plants and fungi of the Glomeromycota is regulated by phytohormones. The role of jasmonate (JA) in AM colonization has been investigated in the dicotyledons Medicago truncatula, tomato and Nicotiana attenuata and contradicting results have been obtained with respect to a neutral, promotive or inhibitory effect of JA on AM colonization. Furthermore, it is currently unknown whether JA plays a role in AM colonization of monocotyledonous roots. Therefore we examined whether JA biosynthesis is required for AM colonization of the monocot rice. To this end we employed the rice mutant constitutive photomorphogenesis 2 (cpm2), which is deficient in JA biosynthesis. Through a time course experiment the amount and morphology of fungal colonization did not differ between wild-type and cpm2 roots. Furthermore, no significant difference in the expression of AM marker genes was detected between wild type and cpm2. However, treatment of wild-type roots with 50 μM JA lead to a decrease of AM colonization and this was correlated with induction of the defense gene PR4. These results indicate that JA is not required for AM colonization of rice but high levels of JA in the roots suppress AM development likely through the induction of defense.

Nymphicidal effect of vegetal extracts of Annona mucosa and Anonna crassiflora (Magnoliales, Annonaceae) against rice stalk stink bug, Tibraca limbativentris (Hemiptera, Pentatomidae)

This study aimed to verify the chloroform-methanol nymphicidal action of extracts of Annona mucosa leaves and seeds and of A. crassiflora seeds on second instar nymphs of rice stalk stink bug, Tibraca limbativentris. For each extract the concentrations of 0.5%, 1.0%, 2.0%, 4.0%, 8.0%, and two control treatments (water and Tween80®) were used. The results show that the seed extracts of A. mucosa and A. crassiflora have insecticidal activity against the T. limbativentris nymphs with statistical significance for all concentrations when compared with controls. The seed extract of A. mucosa showed the higher toxicity with greater than 75% mortality at a concentration of 1.0% in the first 24 h after application. The leaf extract of A. mucosa presented the lowest toxicity with no more than 40% mortality. The seed extract of A. crassiflora showed intermediate toxicity among all the tested extracts, and the nymph's mortality exceeded 80% for the highest concentration after 120 h of application. Considering these results, we were able to observe that the seeds extract of A. mucosa may be an alternative for the control of bed bug nymphs T. limbatriventris, especially for small producers.

Molecular and pathogenic variability of Drechslera isolates from oats

Severe epidemics of leaf blotch and black leaf spot of oat (Avena sativa) caused by Drechslera avenae and Drechslera sp., respectively, are frequently observed in the State of Paraná, Brazil. Although some morphological differences between the isolates causing two different symptoms were noticed, the genetic relationship between them was not clear. Twenty-four isolates of D. avenae and Drechslera sp, collected between 1996-98, were assessed for the genetic variability by molecular and pathogenic analyses. The amplification products using primer pair ITS4/ITS5 showed a fragment length of approximately 600 bp for all the isolates except for one black spot isolate, where the fragment length was approximately 550 bp. Restriction enzymes Hinf I and Taq I, that cut in the ITS region, produced similar restriction patterns for all the isolates, whereas four others produced variable restriction patterns. RAPD analysis also showed distinctive patterns for some isolates. No clear difference between the black spot and the leaf blotch isolates was observed either by the molecular or by the pathogenicity analysis. Nonetheless, the rDNA analysis suggests that Drechslera probably comprises at least three distinct taxa. The results indicate that the difference observed between the isolates originating from two types of symptoms is due to intra-specific variants of D. avenae.

Garlic viral complex: identification of Potyviruses and Carlavirus in Central Brazil

Garlic viruses often occur in complex infections in nature. In this study, a garlic virus complex, collected in fields in Brazil, was purified. RT-PCR was performed using specific primers designed from the consensus regions of the coat protein genes of Onion yellow dwarf virus, a garlic strain (OYDV-G) and Leek yellow stripe virus (LYSV). cDNA of Garlic common latent virus (GCLV) was synthesized using oligo-dT and random primers. By these procedures individual garlic virus genomes were isolated and sequenced. The nucleotide sequence analysis associated with serological data reveals the presence of two Potyvirus OYDV-G and LYSV, and GCLV, a Carlavirus, simultaneously infecting garlic plants. Deduced amino acid sequences of the Brazilian isolates were compared with related viruses reported in different geographical regions of the world. The analysis showed closed relations considering the Brazilian isolates of OYDV-G and GCLV, and large divergence considering LYSV isolate. The detection of these virus species was confirmed by specific reactions observed when coat protein genes of the Brazilian isolates were used as probes in dot-blot and Southern blot hybridization assays. In field natural viral re-infection of virus-free garlic was evaluated.

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.

Resistance spectra of wheat cultivars and virulence diversity of Magnaporthe grisea isolates in Brazil

Seventy-two monoconidial isolates of Magnaporthe grisea were obtained from the States of Mato Grosso do Sul and Paraná. The isolates were inoculated on seedlings of 20 wheat (Triticum aestivum) cultivars under greenhouse conditions. The virulence diversity of M. grisea was assessed based on compatible and incompatible reactions of leaf blast on wheat cultivars. Fifty-four distinct virulence patterns were identified on test cultivars among the isolates collected from the two wheat growing States. Sixteen of these isolates corresponding to 22.2% showed similar virulence pattern. None of the wheat cultivars was resistant to all isolates of M. grisea, but the cultivars differed in degree of resistance as measured by the relative spectrum of resistance (RSR) and disease index (DI). Among the cultivars the RSR ranged from 0 to 53.3% and DI from 0.4662 to 0.9662 (0 to 1 scale). The wheat cultivar BR18 exhibited a broad resistance spectrum in relation to the rest of the tested cultivars to the isolates of M. grisea, and can be used in wheat resistance breeding.

Potential of non-pathogenic Fusarium oxysporum isolates for control of Fusarium wilt of tomato

This study was done to evaluate the efficiency of non-pathogenic Fusarium oxysporum isolates (141/3, 233, 233/1, 245, 245/1, 251, 251/2, 251/5, and 257) in controlling vascular wilt caused by F. oxysporum f. sp. lycopersici, race 2 (isolates C-21A, TO11, and TO245) in tomato (Lycopersicon esculentum) cv. Viradoro seedlings. In order to determine the effect of non-pathogenic F. oxysporum isolates in tomato plants, the root system of 30-day-old seedlings was immersed in conidial suspensions (10(6) ml-1) of each isolate and the seedlings were transplanted to a cultivation substrate. Thirty-five days after transplanting it was observed that the non-pathogenic F. oxysporum isolates were not pathogenic to the cv. Viradoro nor did they affect seedling development. The efficiency of the non-pathogenic F. oxysporum isolates in controlling Fusarium wilt was determined by immersing the tomato seedling roots in the conidial suspension (10(6) ml-1) of each isolate and then transplanting them into substrates previously infested with isolates of F. oxysporum f.sp. lycopersici, race 2 (10(5) conidia ml-1 of substrate). Evaluations were performed 35 days after transplanting, for severity in scale with 1=healthy plant to 6=dead plant or plant showing vessel browning and wilted leaves up to the leader shoot and seedling height. The non-pathogenic F. oxysporum isolates were efficient in reducing the severity of the disease and maintaining normal plant development. These results provide evidence of the antagonistic activity of non-pathogenic F. oxysporum isolates in controlling vascular wilt caused by F. oxysporum f. sp. lycopersici race 2 in tomato.

Citrus exocortis viroid and Hop Stunt viroid Doubly infecting grapevines in Brazil

Viroids, non-protein-coding small (246-401 nt) circular single-stranded RNAs with autonomous replication, are currently classified into two families. Within the family Pospiviroidae, Citrus exocortis viroid (CEVd) belongs to the genus Pospiviroid while Hop stunt viroid (HSVd) is the single member of the genus Hostuviroid. These pathogens are distributed worldwide and infect a large number of hosts. In Brazil, isolates of CEVd and HSVd have been detected in both citrus and grapevine. To characterize and study the genetic variability of these viroids, total RNA from leaves of grapevine Vitis vinifera 'Cabernet Sauvignon' and V. labrusca 'Niagara Rosada' from Bento Gonçalves, RS, was used as a template for RT-PCR amplification with specific primers for the five viroids described infecting grapevines [HSVd, CEVd, Grapevine yellow speckle viroid 1 (GYSVd-1), Grapevine yellow speckle viroid 2 (GYSVd-2) and Australian grapevine viroid (AGVd)]. Leaf samples of Citrus medica infected with CEVd from São Paulo were also analyzed. The resulting products were separated by agarose gel electrophoresis and DNA fragments of the expected size were eluted, cloned and sequenced. The grapevine samples analyzed were doubly infected by CEVd and HSVd. A phylogenetic analysis showed that the Brazilian grapevine HSVd variants clustered with other grapevine HSVd variants, forming a specific group separated from citrus variants, whereas the Brazilian CEVd variants clustered with other citrus and grapevine variants.

PCR amplification and sequence analyses of reverse transcriptase-like genes in Crinipellis perniciosa isolates

Reverse transcriptase (RT) sequence analysis is an important technique used to detect the presence of transposable elements in a genome. Putative RT sequences were analyzed in the genome of the pathogenic fungus C. perniciosa, the causal agent of witches' broom disease of cocoa. A 394 bp fragment was amplified from genomic DNA of different isolates of C. perniciosa belonging to C-, L-, and S-biotypes and collected from various geographical areas. The cleavage of PCR products with restriction enzymes and the sequencing of various RT fragments indicated the presence of several sequences showing transition events (G:C to A:T). Southern blot analysis revealed high copy numbers of RT signals, forming different patterns among C-, S-, and L-biotype isolates. Sequence comparisons of the predicted RT peptide indicate a close relationship with the RT protein from thegypsy family of LTR-retrotransposons. The possible role of these retrotransposons in generating genetic variability in the homothallic C. perniciosa is discussed.

Characterization of Xanthomonas axonopodis pv. phaseoli isolates

A simple, quick and easy protocol was standardized for extraction of total DNA of the bacteria Xanthomonas axonopodis pv. phaseoli. The DNA obtained by this method had high quality and the quantity was enough for the Random Amplified Polymorphic DNA (RAPD) reactions with random primers, and Polymerase Chain Reaction (PCR) with primers of the hypersensitivity and pathogenicity gene (hrp). The DNA obtained was free of contamination by proteins or carbohydrates. The ratio 260nm/380nm of the DNA extracted ranged from 1.7 to 1.8. The hrp gene cluster is required by bacterial plant pathogen to produce symptoms on susceptible hosts and hypersensitive reaction on resistant hosts. This gene has been found in different bacteria as well as in Xanthomonas campestris pv. vesicatoria (9). The primers RST21 and RST22 (9) were used to amplify the hrp gene of nine different isolates of Xanthomonas axonopodis pv. phaseoli from Botucatu, São Paulo State, Brazil, and one isolate, "Davis". PCR amplified products were obtained in all isolates pathogenic to beans.

Differentiation of Colletotrichum gloeosporioides isolates by using total proteins and esterase electrophoretic patterns and extracellular enzymes production

Isolates of Colletotrichum gloeosporioides (ISO-1, ISO-2, ISO-3, ISO-4, ISO-5 and ISO-6), the causal agent of anthracnose disease on mango fruits, were characterized by electrophoretic patterns of total proteins and esterase in polyacrylamida gel, and also, by production of extracellular enzymes on specific solid substrate. The electrophoretic analysis showed variation in number, intensity of coloration and position of the bands in the gel at each studied system tested. In contrast to the monomorphic behavior to total proteins, high esterase polymorfism was observed indicating difference among isolates. All isolates showed the activity of extracellular enzymes such as amylase, lipase, and protease with some variation among them. The proteolitic activity seemed to be more accentuated than the two other enzymes studied.

In vitro sensitivity of Fusarium graminearum isolates to fungicides

Head blight of wheat is a disease of global importance. In Brazil, it can cause damage of up to 27%. As resistant cultivars are not available yet, short-term disease control relies on the use of fungicides. The first step to reach effective management is to identify potent fungicides. In vitro experiments were conducted to determine the inhibitory concentration 50% (IC50) for mycelial growth or conidial germination, according to the chemical group of fungicides, of five Fusarium graminearum isolates of different origins. The following demethylation inhibitor (DMI) fungicides were tested: epoxiconazole, cyproconazole, metconazole, prochloraz, protioconazole and tebuconazole. In addition, azoxystrobin, kresoxim-methyl, pyraclostrobin and trifloxystrobin were included in the study, representing Quinone outside inhibitor fungicides (QoI), as well as a tubulin synthesis inhibitor, carbendazim and two ready mixtures, trifloxystrobin + tebuconazole or trifloxistrobin + prothioconazole. DMI's showed lower IC50 values compared to the QoI's. For the five tested isolates, in the overall mean, IC50 considering mycelial growth ranged for DMI's from 0.01 mg/L (metconazole, prochloraz and prothioconazole) to 0.12 mg/L (cyproconazole) and considering conidial germination for QoI's from 0.21 mg/L (azoxystrobin) to 1.33 mg/L (trifloxystrobin). The IC50 for carbendazim was 0.07 mg/L. All tested isolates can be considered sensitive to the studied DMI's, although certain differences in sensitivity could be detected between the isolates originating from one same state.

In vitro antimicrobial activity of aqueous extracts from Lentinula edodes isolates against Colletotrichum sublineolum and Xanthomonas axonopodis pv. Passiflorae

The aim of this study was to evaluate the antimicrobial activity of aqueous extracts from fruiting bodies of different isolates of Lentinula edodeson the pathogens Colletotrichum sublineolum, the causal agent of anthracnose in sorghum, and Xanthomonas axonopodispv. passiflorae, the causal agent of bacterial spot in passion fruit. Results showed that the aqueous extracts from isolates LE JAB-K and LE 95/01 significantly reduced C. sublineolumspore germination,while the isolate LE 96/22 was the only one to inhibit the pathogen mycelial growth. However, all L. edodesisolates showed inhibitory effect on C. sublineolumappressorium formation. Regarding X. axonopodispv. passiflorae, the aqueous extracts from all L. edodesisolates significantly reduced the in vitromultiplication of the bacterium. However, antimicrobial activity was lost when the extracts were autoclaved, demonstrating their thermolabile property. The aqueous extract from isolate LE 96/22 was also partially purified by anion exchange chromatography and fraction V exhibited high inhibitory activity on the in vitromycelial growth of C. sublineolum, while the multiplication of X. axonopodispv. passifloraewas inhibited by fractions IV, V and VII. Thus, L. edodesisolates were shown to produce compounds exhibiting antifungal and antibacterial activities against phytopathogens, which are mainly concentrated in fraction V.

Comparative pathogenicity of bovine herpesvirus 1 (BHV-1) subtypes 1 (BHV-1.1) and 2a (BHV-1.2a)

The study aimed to examine the capacity of two bovine herpesvirus type 1 (BHV-1) isolates of different subtypes (EVI 123/96, BHV-1.1; SV265/98, BHV-1.2a) to induce respiratory disease in calves. These two isolates are representative of the BHV-1 subtypes prevalent in Brazil. Viral subtypes were confirmed by monoclonal antibody analysis and by restriction enzyme digestion of viral genomes. The viruses were inoculated intranasally into seven 3 months old calves (four with BHV-1.1, three with BHV-1.2a). Three other calves of identical age and condition were kept as uninfected controls. In both groups of infected calves, the clinical signs observed were consistent with typical infectious bovine rhinothracheitis (IBR), including pyrexia, apathy, anorexia, nasal and ocular mucopurulent discharges, erosions on the nasal mucosa, conjunctivitis, lachrymation, redness of nasal mucosa, dyspnoea, coughing, tracheal stridor and enlargement of retropharingeal, submandibular and cervical lymphnodes. No significant differences were observed between the clinical scores attributed to both groups. Virus shedding in nasal and ocular secretions were also similar, apart from a significant difference in nasal virus shedding on day 1 to 3 post-inoculation, which was higher for BHV-1.1 than for BHV-1.2a. Following corticosteroid induced reactivation of the latent infection, recrudescence of clinical signs was also observed, with no significant differences on both groups. It was concluded that both subtypes BHV-1.1 and BHV-1.2a were able to induce clinically undistinguishable respiratory disease in calves, either subsequent to a primary infection or following reactivation.

Phylogenetic and pathotype analysis of Escherichia coli swine isolates from Southern Brazil

The current study evaluated the presence of virulence factors by a multiplex PCR technique and then phylogenetically classified the studied strains into groups A, B1, B2 and D, according to Clermont et al. (2000), in 152 intestinal and extraintestinal swine isolates of Escherichia coli. Seventy seven isolates tested were positive for virulence factors. Phylogenetic characterization placed 21 samples into group A, 65 into B1, 19 into B2 and 47 into D. Fourteen urine samples were classified as uropathogenic E. coli (UPEC), nine were both UPEC and enterotoxigenic E. coli (ETEC) and four were ETEC only. The most common phylogenetic classifications were B1 and D groups. Of the analyzed fecal samples, 25 were classified as ETEC. Phylogenetically, the group of higher occurrence was B1, followed by B2, A and D. For the small intestine samples, 20 were classified as ETEC. Phylogenetic analysis found groups B1 and A to be the most commons in these samples. Six isolated tissue samples were classified as ETEC and most of them were designated as group D by phylogenetic classification. The phylogenetic analysis could be employed in veterinary laboratories in the E. coli isolates screening, including the possibility of vaccine strain selection and epidemiological searches.