Luiz A. Maffia

Suppression of Botrytis cinerea sporulation by Clonostachys rosea on rose debris: a valuable component in Botrytis blight management in commercial greenhouses

Abstract Botrytis blight, caused by Botrytis cinerea (Bc), is an important disease on roses grown in plastic greenhouses in Brazil. Biocontrol with Clonostachys rosea (Cr) applied to leaves and crop debris to reduce pathogen sporulation can complement other control measures for disease management. Two experiments, each with a rose cultivar, were conducted in a plastic greenhouse. For ‘Red Success,’ four treatments were compared: (1) control; (2) fortnightly sprays of Cr; (3) weekly sprays of mancozeb; and (4) weekly sprays of either Cr or mancozeb to the lower third of the plants and the debris. For ‘Sonia,’ treatment 4 was not included. Samples were taken from debris (leaves and petals) at ten 15-day intervals and plated on PCA medium. Sporulation of fungi and incidence of Botrytis blight on buds were assessed. For both cultivars, C treatments significantly ( P =0.05) reduced Bc sporulation. However, disease incidence was not consistently reduced, probably because the applications of C. rosea started when Botrytis blight epidemic was advanced and no sanitation practices were performed on nontreated plots. From the present and previous studies, continuous application of Cr on debris, associated with sanitation practices, has the potential to reduce Bc sporulation and disease incidence in the buds.

Escala de notas para quantificação da ferrugem em Eucalyptus

Based on the pustule size, a rating scale was developed for evaluation of rust severity in inoculated seedlings of Eucalyptus sp. as follow: S0 = immunity or hypersensitive reaction, with necrosis or fleck; S1 = small pustules, 1.6 mm diameter. This scale was checked in a segregant progeny of E. grandis by using the RAPD AT9/917 marker, tightly linked to a rust resistance locus. Only plants classified as S0 and S1 had the RAPD marker and were considered resistant. The error in the classification of resistant and susceptible plants was low (8%). The use of this scale allowed a very fast and accurate screening of rust resistant genotypes.

Characterization of Isolates of Phytophthora infestans from Southern and Southeastern Brazil from 1998 to 2000

The population of Phytophthora infestans in Brazil was first characterized 12 years ago. In this research, isolates of P. infestans from potato (n = 184) and tomato (n = 267) collected in southern and southeastern Brazil were characterized to provide more detailed analysis of the current structure of the population. All 451 isolates were analyzed for mating type, and subsets of the isolates were analyzed for allozymes, restriction fragment length polymorphism fingerprint, mtDNA haplotypes, and metalaxyl resistance. Tomato isolates were all of A1 mating type, mtDNA Ib, and US-1 genotype or some variant within this clonal lineage. Of the potato isolates, 82% were A2 mating type, mtDNA IIa, BR-1 genotype, which is a new lineage of P. infestans. All A2 isolates were found on potato, whereas 91% of the A1 isolates were from tomato. A1 and A2 isolates were never found in the same field. The frequency of resistance to metalaxyl was higher in isolates from tomato (55%) than in isolates from potato (38%). After more than a decade of coexistence of isolates of the A1 and A2 mating types, the population was highly clonal, dominated by the BR-1 and US-1 clonal lineages.

Myrtaceae species resistance to rust caused by Puccinia psidii

Seeds from different species and provenances of Myrtaceae, collected from wild populations in Australia, were screened for resistance to rust caused by Puccini psidii. Seedlings were inoculated with a suspension of rust inoculum and incubated in a mist chamber in the dark for 24 h. Subsequently, the plants were transferred to a growth chamber and rust reaction was evaluated 12 days later. Inter- and intra-specific variability was observed among and within the myrtaceae species. Independent of the provenance, the most resistant species were: Corymbia calophylla ‘rosea’, C. tesselaris, Melaleuca ericifolia, Eucalyptus tereticornis, E. resinifera, E. scias subsp. scias, E. paniculata, E. pellita and C. intermediata. In contrast, M. nesophila, M. alternifolia, M. cajuputi subsp. cajuputi, M. leucadendra, M. quinquenervia, E. cloeziana, E. diversicolor, E. regnans and E. grandis displayed the highest number of susceptible plants. Among those additional myrtaceaceous genera which were tested for their reaction to rust the most resistant were Asteromyrtus dulcia, A. tenuifolia, Gossia fragrantissima, Lophostemon confertus, Syzygium australe, S. wilsonii subsp. cryptophlebium, Archirhodomytus beckleri, Acmena smithii and Syzygium alatoramulum. Pericalymma ellipticum, Kunzea baxteri, Astartea heteranthera, Regelia ciliata, Rhodomyrtus psidioides and Syncarpia glomulifera were the most susceptible species.

Rhizobacterial promotion of eucalypt rooting and growth

A total of 107 rhizobacterial isolates, obtained from the rhizosphere of eucalypt clones were tested as rooting inducers of cuttings and mini-cuttings planted in substrate composed of carbonized rice husk and vermiculite (1:1). Cuttings and mini-cuttings were planted in conical plastic tubes containing treated and untreated (control) substrate and kept under intermittent mist irrigation at 26-28oC. After 35 days, rooting percentage and dry root matter of cuttings were evaluated. Ten isolates capable of providing gains of up to 110% in root formation and up to 250% in root biomass over non-inoculated control cuttings were selected. Gains in rooting varied according to clone and isolate tested. The greatest gains were obtained for the mini-cuttings exhibiting the lowest rooting efficiency. Among the ten isolates tested, only 3918 (code R98) and MF4 (code R87), produced 3-indole-acetic acid in vitro, at concentrations of 0.7 and 0.67 µg ml-1, respectively. Significant increases in rooting and root dry matter of cuttings grown on rhizobacteria-inoculated substrate were found when compared to untreated or indole-butyric acid (IBA) treated mini-cuttings.

Avaliação de produtos alternativos para controle da requeima do tomateiro

The efficacy of alternative products to manage tomato (Lycopersicon esculentum) late blight, caused by Phytophthora infestans, was evaluated in three field trials (E) that compared: E1- [chili pepper (Capsicum chinense) + black pepper (Piper nigrum) + clove (Syzygium aromaticum) + turmeric (Curcuma longa) + garlic (Allium sativum) extracts]; (black pepper + clove + garlic extracts); and (clove + turmeric + garlic extracts); E2 - neem (Azadirachta indica) oil (0.5%), crude cow milk diluted in water (20% v/v), and Bordeaux mixture; E3 - homeopathic preparation (from tomato tissue infected with P. infestans - C30), the water-ethanol mixture, and Bordeaux mixture. All experiments had two controls: no sprays and metalaxyl. Severity at halfway through the epidemic (Y50); at the end of the epidemic (Ymax); area under disease progress curve (AUDPC); and disease progress rate (r) were estimated. None of the extracts reduced Y50, Ymax, AUDPC, or r values. Neem oil and Bordeaux mixture resulted in similar Y50 values (3% and 1%, respectively). Ymax (44%) in plots treated with neem was higher than in those treated with Bordeaux mixture (14%). Milk at 20% did not reduce Ymax. Values of r (0.161) and AUDPC (533) were lower with neem oil than in control (r = 0.211 and AUDPC = 1186) and similar to the Bordeaux mixture plots (r = 0.156 and AUDPC = 130). Values of r and AUDPC on plots treated with milk were similar to those in the control plots. There was no significant reduction of Y50, Ymax, AUDPC, or r values when plants were treated with homeopathic product. Bordeaux mixture was the most efficient treatment in controlling late blight. Neem oil is potentially useful. Integrated management must be implemented to keep late blight at acceptable levels on alternative tomato production systems.

Biological control ofBotrytis cinerea in residues and flowers of Rose (Rosa hybrida)

Microbial isolates from living petals, petal residues and leaf residues of rose, and from laboratory collections, were evaluated for control ofBotrytis cinerea in rose. In leaf residues artificially infested withB. cinerea, isolates of the filamentous fungiGliocladium roseum, FR136 (unidentified) andTrichoderma inhamatum reduced sporulation of the pathogen by >90%, other filamentous fungi were 25–90% effective, and those of yeasts and bacteria were <50% effective. In artificially inoculated petal residues, no microbe reduced sporulation ofB. cinerea by >75%, but isolates ofCladosporium oxysporum and four yeasts were 51–75% effective, and three filamentous fungi, eight yeasts andBacillus subtilis isolates were 26–50% effective. Isolates ofT. inhamatum, C. oxysporum andG. roseum performed best againstB. cinerea among isolates evaluated in leaf residues naturally infested with the pathogen and indigenous microorganisms. Totals of ten isolates of filamentous fungi (includingC. oxysporum andC. cladosporioides), two of yeasts and five ofBacillus subtilis completely prevented lesion production byB. cinerea in detached petals, and a further six isolates of filamentous fungi (includingG. roseum) and six yeasts were 90–99% effective. Isolates ofC. oxysporum, C. cladosporioides andB. subtilis, the most effective microorganisms againstB. cinerea in flower buds, reduced number of lesions in the range of 42–65% compared with 59–89% for à standard fungicide (vinclozolin). It is suggested that application of leading antagonists Jo living rose leaves and flowers should optimize control of inoculum production byB. cinerea when the tissues die. Optimal biocontrol of lesion production in flower buds requires a better understanding of the microenvironment of petals.

Development ofClonostachys rosea and interactions withBotrytis cinerea in rose leaves and residues

The effect of microclimate variables on development ofClonostachys rosea and biocontrol ofBotrytis cinerea was investigated on rose leaves and crop residues. C.rosea established and sporulated abundantly on inoculated leaflets incubated for 7–35 days at 10°, 20° and 30°C and then placed on paraquat—chloramphenical agar (PCA) for 15 days at 20°C. On leaflets kept at 10°C, the sporulation after incubation on PCA increased from 60% to 93% on samples taken 7 to 21 days after inoculation, but decreased to 45% on material sampled after 35 days. A similar pattern was observed on leaves incubated at either 20° or 30°C. The sporulation ofC. rosea on leaf disks on PCA was not affected when the onset of high humidity occurred 0, 4, 8, 12 or 16 h after inoculation. However, sporulation was reduced to 54–58% on leaflets kept for 20–24 h under dry conditions after inoculation and before being placed on PCA. The fungus sporulated on 68–74% of the surface of leaf disks kept for up to 24 h at high humidity after inoculation, but decreased to 40–51% if the high humidity period before transferral to PCA was prolonged to 36–48 h. The growth ofC. rosea on leaflets was reduced at low inoculum concentrations (103 and 104 conidia/ml) because of competition with indigenous microorganisms, but at higher concentrations (105 and 106 conidia/ml) the indigenous fungi were inhibited. Regardless of the time of application ofC. rosea in relation toB. cinerea, the pathogen’s sporulation was reduced by more than 99%. The antagonist was able to parasitize hyphae and conidiophores ofB. cinerea in the leaf residues. AsC. rosea exhibited flexibility in association with rose leaves under a wide range of microclimatic conditions, and in reducingB. cinerea sporulation on rose leaves and residues, it can be expected to suppress the pathogen effectively in rose production systems.

Molecular Diversity and Evolutionary Processes of Alternaria solani in Brazil Inferred Using Genealogical and Coalescent Approaches

Alternaria spp. form a heterogeneous group of saprophytic and plant-pathogenic fungi widespread in temperate and tropical regions. However, the relationship between evolutionary processes and genetic diversity with epidemics is unknown for several plant-pathogenic Alternaria spp. The interaction of Alternaria solani populations with potato and tomato plants is an interesting case study for addressing questions related to molecular evolution of an asexual fungus. Gene genealogies based on the coalescent process were used to infer evolutionary processes that shape the A. solani population. Sequences of the rDNA internal transcribed spacer (ITS) region and the genes which encode the allergenic protein alt a 1 (Alt a 1) and glyceraldehyde-3-phosphate dehydrogenase (Gpd) were used to estimate haplotype and nucleotide diversity as well as for the coalescent analyses. The highest number of parsimony informative sites (n = 14), nucleotide diversity (0.007), and the average number of nucleotide differences (3.20) were obtained for Alt a 1. Although the highest number of haplotypes (n = 7) was generated for ITS, haplotype diversity was the lowest (0.148) for this region. Recombination was not detected. Subdivision was inferred from populations associated with hosts but there was no evidence of geographic subdivision, and gene flow is occurring among subpopulations. In the analysis of the Alt a 1, balancing selection and population expansion or purifying selection could have occurred in A. solani subpopulations associated with potato and tomato plants, respectively. There is strong evidence that the subpopulation of A. solani that causes early blight in potato is genetically distinct from the subpopulation that causes early blight in tomato. The population of A. solani is clonal, and gene flow and mutation are the main evolutionary processes shaping its genetic structure.

Efeito de Secreções da Glândula Mandibular de Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae) Sobre a Germinação de Conídios de Botrytis cinerea Pers. Fr.

This research investigated the effect of mandibular gland secretions of Atta sexdens rubropilosa Forel on the germination of Botrytis cinerea Pers. Fr. conidia. This fungus attacks several species of cultivated plants of economic importance. From 20 mandibular glands of workers of the leaf-cutting ant (3.60-4.30 mm head capsule width) we obtained 9.4 mg of acetone soluble secretion. To this mass, sterile water and the dispersing agent Tween 20 were added to prepare four doses which composed the treatments of 0.94 mg/ml, 4.70 mg/ml, 9.40 mg/ml and 37.60 mg/ml. We also used a negative control (water), a solvent control (acetone) and a positive control (the Mancozeb fungicide at 1600 ppm). Five microliters of B. cinerea conidia suspension were added to excavated plates maintained at 20°C in the dark. Therefore the solution concentrations of the treatments were reduced to half, that is 0.47 mg/ml, 2.35 mg/ml, 4.70 mg/ml and 18.80 mg/ml. A total of 12 replicates were performed for each of four treatments. The percentage of conidia germination was obtained 8h after treatments. Results by Probit analysis of data indicated that higher gland concentrations led to higher inhibition of conidia germination. The concentration of 18.80 mg/ml had an inhibitory effect (94.2%) similar to that of Mancozeb (95.3%).