Morphological and molecular characterization of Colletotrichum spp. from citrus orchards affected by postbloom fruit drop in Brazil

Brazilian isolates of Colletotrichum spp. from citrus orchards affected by postbloom fruit drop were examined for colony colour, mycelial growth, benomyl-resistance, pathogenicity, and genetic variability by random amplified polymorphic DNA (RAPD) analysis. All isolates were obtained from flowers and persistent calyxes from different citrus hosts from São Paulo, Brazil. DNA polymorphisms detected after amplification with random 10-mer primers were used to classify the isolates into two groups. Group I isolates grew rapidly on potato-dextrose agar (PDA) and were sensitive to benomyl, and group II isolates grew slowly on PDA and were benomyl-resistant. Colletotrichum acutatum was analyzed by RAPD and had high genetic similarity with group II isolates of Colletotrichum from citrus. Probably, the group I is C, gloeosporioides and group II is C. acutatum.

Activity of benomyl for control of postbloom fruit drop of citrus caused by Colletotrichum acutatum

Postbloom fruit drop (PFD) of citrus caused by Colletotrichum acutatum produces orange-brown lesions on petals and induces the abscission of young fruitlets and the retention of the calyces. Despite the fact that C. acutatum is not highly sensitive to benomyl in culture, this fungicide provides good control of the disease under field conditions. This study was undertaken to determine the effect of benomyl on various stages of disease development to understand the basis for its effectiveness in the field. We found that benomyl at 1.0 μg/ml reduced colony area of C. acutatum by about 75% and completely inhibited growth of C. gloeosporioides. Benomyl did not prevent conidial germination even at 100 μg/ml, but reduced germ tube elongation at 10 and 100 μg/ml. When benomyl was applied to flower clusters on screen-house-grown plants before inoculation, disease severity was greatly reduced. Applications at 24 and 48 h, but not at 72 h, after inoculation reduced PFD severity. Application of benomyl to symptomatic petals not bearing conidia did not prevent or reduce production of inoculum. Application to petals bearing conidia reduced viability of these fungal propagules by only about 50%. The viability of appressoria on mature leaves was not affected by benomyl application. Even when appressoria on mature leaves were stimulated to germinate by treatment with flower extracts, subsequent application of benomyl did not reduce propagule numbers below original levels. Benomyl appears to act by preventing infection and early development of the fungus in petals. However, once symptoms have developed, this fungicide has only minimal effects on further disease development and spread.

Evaluation of systems for timing of fungicide sprays for control of postbloom fruit drop of citrus in Brazil

Postbloom fruit drop (PFD) of citrus, caused by Colletotrichum acutatum, infects petals of citrus flowers and produces orange-brown lesions that induce the abscission of young fruitlets and the retention of calyces. Proper timing of fungicide applications is essential for good disease control. Different systems for timing of fungicide applications for control of PFD in a major citrus-growing region in southern São Paulo state in Brazil were evaluated from 1999 to 2002. The following programs were compared to an unsprayed control using counts of diseased flowers, persistent calyces, or fruit: (i) a phenology-based program currently recommended in Brazil with one application at early and another at peak bloom; (ii) the Florida PFD model; (iii) the postbloom fruit drop-fungicide application decision system (PFD-FAD), a new computer-assisted decision method; and (iv) grower's choice. In 1999, no disease developed, sprays applied with the phenology-based program had no effect, and the Florida PFD model saved two sprays compared with the phenology-based program. In 2000, PFD was moderate and the phenology-based and growers' choice treatments had a significantly lower number of persistent calyces and higher fruit numbers than the control, but no differences were found between those treatments and the PFD model. In 2001, PFD was severe with considerable yield loss. The PFD model, the phenology-based program, and the grower's choice reduced flower blight and the number of persistent calyces, and improved fruit yields with two to three applications, but the PFD-FAD achieved comparable yields with only one spray. In 2002, the disease was mild, with no yield loss, and the Florida PFD model and the PFD-FAD saved one spray compared with the other systems. The PFD model and the PFD-FAD were equally effective for timing fungicide applications to control PFD in Brazil. Scouting of trees is simpler with PFD-FAD; therefore, this system is recommended and should eliminate unnecessary sprays and reduce costs for growers.

Evaluation of fungicide applications to sweet orange at different flowering stages for control of postbloom fruit drop caused by Colletotrichum acutatum

Postbloom fruit drop (PFD), caused by Colletotrichum acutatum, produces blossom blight, fruit abscission and persistent calyces. in groves of Pera-Rio and Natal sweet orange located in Santa Cruz do Rio Pardo and Rincao, São Paulo, Brazil, four experiments were carried out to evaluate the effectiveness of fungicides sprayed alone or as mixtures, at different flowering stages for the control of PFD of citrus. The number of symptomatic flowers, the percentage of fruit set (FS), and the relationship between persistent calyces and total fruit weight per plant were evaluated. The fungicides carbendazim and folpet were sprayed at 0.50 ml and 1.25 ga.i. l(-1) of water, respectively, were superior by all the criteria to the other treatments. Carbendazim and folpet fungicides performed best when they were applied at the green bud through hollow ball stages. Difenoconazole, independent of application timing, was less effective by all criteria used. Application of mancozeb at 1.60 ga.i. l(-1) at the green bud stage followed by application of mancozeb in a tank mix with carbendazim or folpet at 1.0 ml and 1.25 g a.i. l(-1), respectively, during green bud bloom and hollow ball stages were effective for disease control. Carbendazim combined with 0.25% KNO3, reduced the number of persistent calyces and increased fruit production significantly. Applications must be made between green bud and hollow ball stages for best control. Applications only at hollow ball or open flower stages did not provide effective disease control. (C)2007 Elsevier Ltd. All rights reserved.

Benomyl sensitivity of isolates of Colletotrichum acutatum and C. gloeosporioides from citrus

Postbloom fruit drop (PFD) of citrus, caused by Colletotrichum acutatum, produces orange-brown lesions on petals and results in premature fruit drop and the retention of calyces. C. gloeosporioides is common in groves and causes postharvest anthracnose on fruit. Both diseases are controlled effectively by the fungicide benomyl in research fields and commercial orchards. Highly sensitive and resistant isolates of C. gloeosporioides were found, whereas all isolates of C. acutatum tested were moderately resistant. In preliminary studies conducted in vitro with three isolates of each, mycelial growth of sensitive isolates of C. gloeosporioides was inhibited completely by benomyl (Benlate 50 WP) at 1.0 μg/ml, whereas resistant isolates grew well at 10 μg/ml. Growth of all isolates of C. acutatum was inhibited by about 55% at 0.1 μg/ml and by 80% at 1.0 μg/ml. Spore germination of C. acutatum was inhibited more at 0.1 μg/ml than at 1.0 μg/ml or higher concentrations. In all, 20 isolates of C. acutatum from 17 groves and 20 isolates of C. gloeosporioides from 7 groves were collected from locations with different histories of benomyl usage in São Paulo, Brazil, and Florida, United States. Benomyl at 1.0 μ.g/ml completely inhibited growth of 133 isolates of C. gloeosporioides, with the exception of 7 isolates that were highly resistant to the fungicide, whereas all isolates of C. acutatum were only partially inhibited at 0.1 and 1.0 μg/ml. Analysis of variance indicated that the sensitivity of the isolates of C. acutatum was not affected by benomyl usage or grove of origin, and country of origin had only minor effects. No highly resistant or sensitive isolate of C. acutatum was recovered. Partial sequencing of the β-tubulin gene did not reveal nucleotide substitutions in codons 198 or 200 in C. acutatum that usually are associated with benomyl resistance in other fungi.

Modelo de previsão e controle da podridão floral dos citrus causada por Colletotrichum acutatum

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Controle alternativo de Colletotrichum acutatum agente causal da queda prematura dos frutos cítricos

A queda prematura dos frutos cítricos (QPFC), causada por Colletotrichum acutatum, dados os grandes prejuízos que têm causado aos produtores, constitui-se numa doença de grande importância econômica. O controle da doença é feito predominantemente mediante uso de fungicidas, que eleva o custo de produção e afeta negativamente o meio ambiente. Diante disso, este trabalho teve por objetivo buscar um método alternativo de controle da QPFC, mediante o uso de agentes de biocontrole ou de biofertilizantes. Diferentes concentrações de biofertilizantes (originários de duas fontes distintas e denominados de Bio1 e Bio 2); três isolados de Bacillus subtilis (ACB-69; 72 e 77) e três isolados de Trichoderma spp. (ACB-14; 37 e 39) foram testados, isoladamente ou em combinação, sob condições de laboratório, quanto à capacidade inibitória da germinação de conídios de C. acutatum. Estudaram-se, ainda, a produção de metabólitos termoestáveis por B. subtilis e o efeito sobre a germinação do patógeno. Quinze isolados de B. subtilis foram testados quanto à capacidade de prevenir a infecção por C. acutatum em flores destacadas de lima- ácida 'Tahiti' e, no campo, foram instalados dois experimentos, visando a testar ACBs e biofertilizantes no controle da doença. Verificou-se que o isolado ACB-72 (B. subtilis) e ACB-37 (T. pseudokoningii) foram os que mais inibiram a germinação do patógeno. Quanto à produção de metabólitos termoestáveis, ACB-69 e 77 foram os mais eficientes em produzir substâncias antifúngicas, e em quantidades suficientes para inibirem a germinação do patógeno. A mistura dos quatro isolados de Bacillus (ACBs: 69; 72; 77 e AP3) foi o que apresentou maior porcentagem de inibição (73%). Os biofertilizantes (Bio1 e Bio2), em concentrações acima de 10% e, quando em associação com isolados de Trichoderma spp., promoveram maiores inibições na germinação de C. acutatum. em testes com flores destacadas, verificou-se que, onde foram aplicados os ACBs 69; 76; 74 e 77, as porcentagens de pétalas sem sintomas de infecção por C. acutatum foram de 83; 92; 92 e 97%, respectivamente. Mediante avaliações a campo, verificou-se a potencialidade de B. subtilis e de biofertilizantes em controlar a doença.

Diversidad genética de aislamientos de Bacillus subtilis con potencial para el control biológico de Colletotrichum acutatum y Guignardia citricarpa

O setor citrícola enfrenta sérios problemas representados por doenças de flores e frutos jovens que, além de diminuir a produtividade, depreciam os frutos pelo aspecto que conferem aos mesmos. Tais doenças são representadas, principalmente, pela mancha preta dos frutos cítricos (MPC) e pela queda prematura dos frutos cítricos (QPFC), onde a medida predominante de controle é a pulverização com produtos químicos. Entretanto, os custos financeiros e ambientais de aplicações com tais produtos, aliado às crescentes restrições à presença de resíduos, estão a exigir o estudo de novas alternativas. Entre estas, o controle biológico surge como alternativa importante. Sabendo-se que, o conhecimento da biodiversidade dos seres vivos é importante para determinação de suas funções potenciais, o presente trabalho teve por objetivo estudar a diversidade genética, através de marcadores moleculares AFLP, de 32 isolados de B. subtilis com a finalidade de se encontrar, dentre os mesmos, um (ou mais isolados) que apresentasse maior similaridade com o isolado ACB-69, o qual apresenta potencial para o controle da doença. Diante disso, os resultados obtidos neste trabalho, permitiram concluir que: a) os isolados de B. subtilis estudados agruparam-se no filograma de distância genética, independente da procedência ou do hospedeiro; b) os isolados ACB-69 e ACB-83, com potenciais para o controle da queda prematura dos frutos cítricos, compartilham da mesma ancestralidade, o que pode ser inferido pela metodologia aplicada; c) em termos biológicos; o isolado ACB-83 merece mais estudos quanto à viabilidade de controle de doenças de citros, como a queda prematura dos frutos cítricos e a manha preta dos frutos cítricos, sob condições de campo.

Controle químico de Colletotrichum acutatum agente causal da queda prematura dos frutos cítricos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Podridão floral dos citros: variabilidade, sobrevivência e controle do agente causal, Colletotrichum acutatum

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Caracterização molecular de Colletotrichum acutatum e C. gloeosporioides associados a citros, por marcadores ISSR

Pós-graduação em Agronomia (Produção Vegetal) - FCAV

Saccharomyces cerevisiae: a novel and efficient biological control agent for Colletotrichum acutatum during pre-harvest

In this study, we evaluated the efficiency of six isolates of Saccharomyces cerevisiae in controlling Colletotrichum acutatum, the causal agent of postbloom fruit drop that occur in pre-harvest citrus. We analyzed the mechanisms of action involved in biological control such as: production of antifungal compounds, nutrient competition, detection of killer activity, and production of hydrolytic enzymes of the isolates of S. cerevisiae on C. acutatum and their efficiency in controlling postbloom fruit drop on detached citrus flowers. Our results showed that all six S. cerevisiae isolates produced antifungal compounds, competed for nutrients, inhibited pathogen germination, and produced killer activity and hydrolytic enzymes when in contact with the fungus wall. The isolates were able to control the disease when detached flowers were artificially inoculated, both preventively and curatively. In this work we identified a novel potential biological control agent for C acutatum during pre-harvest. This is the first report of yeast efficiency for the biocontrol of postbloom fruit drop, which represents an important contribution to the field of biocontrol of diseases affecting citrus populations worldwide. (C) 2015 Elsevier GmbH. All rights reserved.

Mapas de zonas de risco de epidemias para doenças dos citros no Estado de São Paulo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Podridão floral dos citros: controle preventivo mediante fungicidas, biorreguladores e fertilizantes químicos

Pós-graduação em Agronomia (Produção Vegetal) - FCAV

Acaricides used in conventional and organic citrus orchard: leprosis and phytoseiid mites population control

The citrus leprosis control in São Paulo state is performed exclusively by acaricides to control the vector mite, Brevipalpus phoenicis, which increases the production costs and may affect the beneficial organism's population. Therefore, the aim of this trial was to evaluate during four seasons, the effects of acaricides recommended to control the mite B. phoenicis in conventional and organic citrus over evolution of citrus leprosis and over phytoseiids' population. The experiment was installed in October of 2003 in a citrus orchard in Reginopolis city, state of São Paulo. The experimental design used randomized blocks, the dosages was expressed as mL c.p./100L of water and the treatments were the following: spirodiclofen (20 mL); cyhexatin (50 mL) (used in rotation), lime sulfur (4,000 mL) and control (without pesticide application). However, the rotation between spirodiclofen and cyhexatin began in September 2006. Prior to that time, only spirodiclofen had been used. Surveys were conducted every 15 days on the B. phoenicis, Iphiseioides zuluagai, and Euseius populations. The control level adopted by the B. phoenicis was 8.3%, and the pesticide applications were conducted using tractor-sprayers. During the 2007-08 seasons, 10 infected fallen fruits per plot were collected and the number of leprosis lesions was quantified by each fruit. By the end of the 2007-08 seasons, the productivity, harvest losses, the disease incidence and severity were evaluated. It was found that the lesions' location over the fruit is more important in determining its drop than the lesions' number. The more intense the mite infestation, the greater is the number of lesions, resulting in increased premature fruit drop. A strategy using acaricides spirodiclofen and cyhexatin in rotation promoted more efficient control of B. phoenicis compared to lime sulfur, resulting in greater productivity, lower fruit losses and severity levels. The lime sulfur applications reduced the mite population incidence below the control level; however it did not prevent the lesions' occurrence. The acaricides applicarevented adverse effects on phytoseiid population because there was a reduction of their density.