Production of caffeine alkaloid by Claviceps sorghi

The ergot disease of sorghum (Sorghum bicolor), caused by the fungus Claviceps sorghi, restricted to the Indian sub-continent, is a disease in which the pathogen infects the florets, colonizing the unfertilized ovaries. Losses are higher in hybrid seed production fields due to a higher susceptibility of male sterile lines. The sclerotia of C. sorghi have never been found to contain alkaloids with a tetracyclic ergoline ring system, which is normal in most ergot pathogens. In this work, we show that sclerotia of C. sorghi contain caffeine alkaloid and the ability to produce it in vitro.

Effect of organic amendments on sclerotial germination, mycelial growth, and Sclerotium rolfsii-induced diseases

The addition of organic residues to soil is an option to control some soil-borne diseases. Benzaldehyde and powders of kudzu (Pueraria lobata), velvetbean (Mucuna deeringiana), and pine-bark (Pinus elliottii and P. taeda) added to soil could reduce certain soil-borne diseases. This study evaluated the effects of benzaldehyde and the dried powders of kudzu, velvetbean, and pine-bark as soil amendments on germination and formation of sclerotia, on mycelial growth of Sclerotium rolfsii, on plant survival, and disease incidence. The data showed that high amounts of benzaldehyde (0.4 ml kg-1 of soil) and velvetbean (100 g kg-1) inhibited S. rolfsii mycelial growth and sclerotium germination. However, low amounts of benzaldehyde (0.1 ml kg-1), kudzu (25 g kg-1), and pine-bark (25 g kg-1) stimulated mycelial growth and sclerotium germination. Kudzu (25-100 g kg-1) and velvetbean (25-100 g kg-1) inhibited the formation of sclerotia. Nevertheless, benzaldehyde at 0.2 and 0.4 ml kg-1 stimulated the formation of sclerotia. Kudzu (50 and 100 g kg-1) and pine-bark (50 g kg-1) favored the colonization of sclerotia by Trichoderma sp. The numbers of soybean (Glycine max) plants were higher and diseased plants were lower than the non-amend soil in the following treatments: kudzu (50 and 100 g kg-1), velvetbean (50 and 100 g kg-1), and pine-bark (50 g kg-1). Disease severity on tomato (Lycopersicon esculentum) plants was low in soil treated with kudzu or velvetbean (30 and 35 g kg-1) and pine-bark (35 g kg-1). Dried powders of kudzu, velvetbean, or pine-bark added to soil can reduce disease by reducing pathogen inoculum.

Detection and transmission of Drechslera avenae from oat seed

Detection, symptoms and symptomless transmission of Drechslera avenae (teleomorph Pyrenophora avenae) from seed were investigated. The present study reported that the D. avenae is frequently found in oat (Avena sativa) seed in Argentina. The prevalence of the seed lot infected was 54%. The incidence of seeds infected by D. avenae in the various seed lots from different regions ranged from 0 to 52% (overall mean of 7%). Besides conidia on conidiophore and immature pseudothecia, D. avenae produced small, spherical to pear-shape picnidia containing tiny conidia, on the seed surface. The pathogen was efficiently transmitted at a rate of 55% from seed to coleoptile tips in symptomless seedlings and at 12.5% to plumules. The importance of the infected seed and its epidemiological role are discussed.

Variability in ribosomal DNA genic and spacer regions in Verticillium dahliae isolates from different hosts

Using PCR-based assays with specific primers for amplification of the ribosomal DNA intergenic spacer region (IGS) and a portion of the mitochondrial DNA small subunit ribosomal RNA gene (mtDNA SSU rRNA), the genetic variability among Verticillium dahliae isolates from olive (Olea europaea) and other host species from Argentina and Brazil was estimated. The derived UPGMA-generated phenograms based upon the restriction fingerprinting data of rDNA IGS products revealed genetic differences, correlating with the host of origin. Isolates infecting olive genetically distinct from those from cocoa (Theobroma cacao) and sunflower (Helianthus annuus). Digestion of mitochondrial DNA SSU rRNA PCR products revealed less variability, distinguishing only one isolate from sunflower. Ribosomal DNA ITS restriction patterns were identical for all isolates of V. dahliae, irrespective of host of origin. These preliminary results may have relevance for Verticillium wilt control practices, possibly reflecting a different evolutionary origin, or reproductive isolation of the pathogen in olive, distinct from populations of other hosts.

Variability of the anthracnose fungus Colletotrichum graminicola in sorghum genotype mixtures

This paper reports partial results obtained on the variability of Colletotrichum graminicola developed in response to the host diversity generated by three-line combination of sorghum (Sorghum bicolor) genotypes. Nine sorghum lines were used in this study: CMSXS210B, CMSXS112B, CMSXS215B, CMSXS221B, CMSXS169R, CMSXS180R, CMSXS182R, CMSXS227R, and CMSXS116R. A total of 39 treatments on mixtures and pure stands of the component lines were evaluated in the field for the development of anthracnose, as a natural epidemic. Samples of the single spore isolates of the pathogen of each treatment indicated a reduction in the phenotypic diversity and an increase in the frequency of more complex races in genotype mixtures in relation to the pure stands of each genotype.

Molecular characterization of Brazilian strains of Xanthomonas campestris pv. viticola by rep-PCR fingerprinting

Bacterial canker of grapevine (Vitis vinifera), caused by Xanthomonas campestris pv. viticola was first detected in Brazil in 1998, affecting grapevines in the São Francisco river basin, state of Pernambuco. The disease was also reported in Juazeiro, Bahia and later in Piauí and Ceará. Due to its limited geographical distribution and relatively recent detection in Brazil, very little is known about the pathogen's biology and diversity. Repetitive DNA based-PCR (rep-PCR) profiles were generated from purified bacterial DNA of 40 field strains of X. campestris pv. viticola, collected between 1998 and 2001 in the states of Pernambuco, Bahia and Piauí. Combined analysis of the PCR patterns obtained with primers REP, ERIC and BOX, showed a high degree of similarity among Brazilian strains and the Indian type strain NCPPB 2475. Similar genomic patterns with several diagnostic bands, present in all strains, could be detected. Fingerprints were distinct from those of strains representing other pathovars and from a yellow non-pathogenic isolate from grape leaves. The polymorphism observed among the Brazilian strains allowed their separation into five subgroups, although with no correlation with cultivar of origin, geographic location or year collected.

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.

First report of Fusarium oxysporum f. sp. lycopersici race 3 on tomato in Brazil

Fusarium wilt, caused by three races of Fusarium oxysporum f. sp. lycopersici, is one of the most important diseases of tomato (Lycopersicon esculentum). Races 1 and 2 are distributed worldwide whereas race 3 has a more limited geographic distribution with no report thus far in Brazil. Seven F. oxysporum isolates were obtained from wilted tomato plants of race 1 and 2-resistant hybrids 'Carmen' and 'Alambra' in Venda Nova do Imigrante (State of Espírito Santo), Brazil. Virulence assays were performed using a set of the race differential cultivars: 'Ponderosa' (susceptible to all races), 'IPA-5' (resistant to race 1), 'Floradade' (resistant to races 1 and 2) and 'BHRS-2,3' (resistant to race 3). All isolates were highly virulent to 'Ponderosa', 'IPA-5' and 'Floradade' and were able to infect only a few plants of 'BHRS-2,3'. An additional virulence test was conducted including the same set of cultivars plus Lycopersicon pennellii 'LA 716'. Identical results were obtained with L. pennellii displaying an extreme (immune-like) resistant response. These results indicated that all seven isolates could be classified as F. oxysporum f. sp. lycopersici race 3. This new Fusarium wilt might became an economically important disease since race 3-resistant cultivars adapted to Brazil are not yet available.

Effect of Fusarium graminearum and infection index on germination and vigor of maize seeds

Pathogens in maize (Zea mays) seeds cause serious problems, such as the loss of their capacity to germinative. The objectives of this study were to identify the optimal period for infection of maize seeds on agar colonized by Fusarium graminearum, when incubated for 4, 8, 16 and 32 h, and to evaluate the effect of the fungus on the germination and vigor of seeds with different infection levels. After the respective incubation periods, the seeds were removed from the culture medium and submitted to the blotter test for 3 min with and without superficial disinfection with 1% solution of sodium hypochlorite. Once the optimal period for seed incubation was identified, seeds from the same sample were again placed on the colonized agar for infection. Germination and vigor tests (accelerated aging and cold test) were performed with a mixture of healthy seeds (placed on PDA medium) and inoculated seeds, resulting in seeds with 0, 20, 40, 60, 80 and 100% rates of infection. The results showed that a period of 32 h was long enough to obtain seeds infected by the pathogen. There were no significant effects of fungal infection on seed germination at any of the infection levels, probably due to the high vigor of the maize seed lot tested. Regarding vigor tests, infection levels differed significantly from the control (0% infection), but there were no significant differences among the infection levels.

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

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

Survival of Botrytis cinerea as mycelium in rose crop debris and as sclerotia in soil

Botrytis blight caused by Botrytis cinerea is an important disease of rose (Rosa hybrida) grown in greenhouses in Brazil. As little is known regarding the disease epidemiology under greenhouse conditions, pathogen survival in crop debris and as sclerotia was evaluated. Polyethylene bags with petals, leaves, or stem sections artificially infected with B. cinerea were mixed with crop debris in rose beds, in a commercial plastic greenhouse. High percentage of plant parts with sporulation was detected until 60 days, then sporulation decreased on petals after 120 days, and sharply decreased on stems or leaves after 90 days. Sporulation on petals continued for 360 days, but was not observed on stems after 150 days or leaves after 240 days. Although the fungus survived longer on petals, stems and leaves are also important inoculum sources because high amounts of both are deposited on beds during cultivation. Survival of sclerotia produced on PDA was also quantified. Sclerotia germination was greater than 75% in the initial 210 days and 50% until 360 days. Sclerotia weight gradually declined but they remained viable for 360 days. Sclerotia were produced on the buried petals, mainly after 90 days of burial, but not on leaves or stems. Germination of these sclerotia gradually decreased after 120 days, but lasted until 360 days. Higher weight loss and lower viability were observed on sclerotia produced on petals than on sclerotia produced in vitro

Evaluation of genetic mixtures of sorghum lines for anthracnose resistance management

The main objective of this work was to evaluate the diversification of sorghum (Sorghum bicolor) populations as a way to manage resistance to the sorghum anthracnose fungus Colletotrichum graminicola. A total of 18 three-way hybrids were obtained by crossing six single cross male-sterile F1 hybrids, derived by crossing A (non restorer sterile cytoplasm) and B (non restorer normal cytoplasm) lines, with three fertile R (restorer) lines, previously evaluated for their differential reaction to the pathogen. Variation in the level of resistance was observed, as indicated by the values of the area under the disease progress curve (AUDPC) obtained for each hybrid. Lines contributed differently to the level of resistance of each hybrid. All hybrids in which CMSXS169R was the male progenitor were classified as highly resistant. Some hybrids had a level of resistance superior to the maximum levels of each line component individually.

Pathogenicity, molecular characterization, and cercosporin content of Brazilian isolates of Cercospora kikuchii

Cercospora kikuchii, involved with the defoliation of soybean (Glycine max) plants, is normally associated with Septoria glycines in late season. Seventy-two isolates from different regions in Brazil, obtained mainly from purple stained seeds, showed phenotypic variation. Cercosporin content and rate of colony growth was higly variable among isolates. A strong correlation between cercosporin content and virulence was identified. Genetic variation among and within populations was evaluated based on 86 RAPD loci. The RAPD analysis clustered all isolates into seven groups. No relationship was observed between RAPD groups and geographic origin or cercosporin content. The sequence of the internal spacer regions (ITS1-5.8S-ITS2) from 13 isolates chosen according to the previous RAPD and clustering analysis showed high similarity (97%-100%) to the GenBank sequences of C. kikuchii (AY266160, AY266161, AY152577 and AF291708). It is clear from this work that Brazilian isolates of C. kikuchii from different geographic regions, are variable in relation to virulence, RAPD profiles and cercosporin content. Cercosporin content could be a good parameter for choosing an adequate isolate for screening resistant or tolerant cultivars. Considering that this pathogen is easily seed-borne, findings are expected to show the same haplotypes in different regions. Migration could be favoured by infected seeds as demonstrated by RAPD analysis.

Virulence pattern of Pyricularia grisea isolates from farmers' fields on newly released upland rice cultivars

The virulence pattern of the isolates of Pyricularia grisea from commercial fields of the upland rice (Oryza sativa) cultivars 'Primavera' and 'BRS Bonança' was analyzed. A hundred and seventy monoconidial isolates of the pathogen virulent to 'Primavera' and 139 to 'BRS Bonança' collected from eight fields, during two years (2001-2003) were tested, under greenhouse conditions, on six newly released rice cultivars. Differences in virulence pattern were observed in pathogenic populations of 'Primavera' and 'BRS Bonança'. Isolates with virulence to improved cultivars were common in samples from farmers' fields in the absence of aloinfection. The virulence frequency of P. grisea isolates collected from 'Primavera'' to cultivars 'BRS Vencedora', 'BRS Colosso', 'BRS Liderança', 'BRS Soberana', 'BRS Curinga' and 'BRS Talento', was high in descending order. On the other hand, in the fungus population of 'BRS BRS Bonança' virulence frequency was high in 'BRS Talento', followed by 'BRS Curinga', 'BRS Vencedora', 'BRS Liderança', 'BRS Colosso' and 'BRS Soberana'. While virulence to 'BRS Talento' was rare among isolates from 'Primavera', it was most frequent in isolates of 'BRS Bonança'. The six improved rice cultivars permitted to differentiating agriculturally important virulences in the pathogen population which can be utilized in selecting breeding lines for specific resistance, in rice blast improvement program.

Injuries caused by citrus leafminer (Phyllocnistis citrella) exacerbate citrus canker (Xanthomonas axonopodis pv. citri) infection

After the introduction of citrus leafminer in São Paulo State, an increase in the number of new plants infected with citrus canker has been observed. The interaction between these two organisms is known, but there is no information about how the leafminer damage intensifies citrus canker incidence and severity. The objectives of this paper were to evaluate the effects of leafminer damage in citrus canker infection and its influence on the monocyclic components of the disease on Citrus limonia. Higher incidence of diseased plants, AUDPC (area under the disease progress curve), disease severity and shorter incubation periods were observed in plants inoculated after insect infestation. These factors explain the association found between the higher citrus canker intensity and the damage caused by the insect and show, albeit partially, the consequences of these changes in the spread of the pathogen under natural conditions of infection.