Sour rot is a major postharvest disease of citrus fruit and is caused by the fungal pathogen Geotrichum citri-aurantii. A lack of chemicals certified for the control of this disease has led to the consideration of alternative methods and strategies, such as the use of yeasts as biocontrol agents. The purpose of the present study was to test the ability of yeasts isolated from leaves, flowers, fruit, and soil, and six Saccharomyces cerevisiae isolates to control citrus sour rot, to assess the mechanisms of action of the yeast isolates that were demonstrated to be effective for biocontrol, and to identify the most effective yeast isolates for the biocontrol of G. citri-aurantii. In in vivo assays, three yeast isolates (ACBL-23, ACBL-44, and ACBL-77) showed a potential for controlling sour rot in citrus fruits, both preventatively and curatively. Most of the eight yeast isolates that were assessed for a mechanism of action did not produce antifungal compounds in an amount sufficient to inhibit the growth of the pathogen. Additionally, nutrient competition among the yeast strains was not found to be a biocontrol strategy. Instead, killer activity and hydrolytic enzyme production were identified as the major mechanisms involved in the biocontrol activity of the yeasts. Isolates ACBL-23, ACBL-44, and ACBL-77, which controlled sour rot most effectively, were identified as Rhodotorula minuta, Candida azyma, and Aureobasidium pullulans, respectively. To our knowledge, this is the first report of the potential of C. azyma as a biological control agent against a postharvest pathogen and its ability to produce a killer toxin.

O trabalho teve por finalidade estudar a potencialidade antagonística de isolados de Bacillus subtilis a Colletotrichum acutatum, agente causal da queda prematura dos frutos cítricos (Citrus spp.) (QPFC), sob condições de laboratório e de campo. Foram estudados 64 isolados de B. subtilis, quatro isolados de Bacillus spp. e um isolado de B. thuringiensis quanto à capacidade de inibir o desenvolvimento do patógeno em cultura pareada e quanto à produção de metabólitos com atividade antimicrobiana. Os isolados mais promissores foram testados em condições de campo para controle da doença. In vitro, todos os isolados de Bacillus spp. inibiram o crescimento de C. acutatum, não havendo diferenças significativas entre eles. Os isolados de Bacillus spp. produziram, in vitro, metabólitos capazes de inibir o crescimento micelial de C. acutatum, os quais mantiveram suas atividades capazes de causar a inibição, após autoclavagem a 120 ºC, durante 20 min. Dentre os sete isolados de B. subtilis testados para o controle da QPFC, em condições naturais, o ACB-69 diferiu da testemunha e de vários outros isolados, porém equiparou-se estatisticamente ao benomyl, proporcionando menor porcentagem de flores com sintomas e maior número médio de frutos efetivos. Ainda, sob condições de campo, isolados de cada uma das espécies Trichoderma viride, T. pseudokoningii e T. aureoviride foram ineficientes, apresentando o mesmo comportamento da testemunha. Em relação aos métodos de avaliação da doença, a porcentagem de flores com sintomas foi mais eficiente do que o número médio de frutos efetivos (NMFE), uma vez que esses resultam do efeito direto do patógeno.

Citrus are vulnerable to the postharvest decay caused by Penicillium digitatum, Penicillium italicum, and Geotrichum citri-aurantii, which are responsible for the green mold, blue mold, and sour rot post-harvest disease, respectively. The widespread economic losses in citriculture caused by these phytopathogens are minimized with the use of synthetic fungicides such as imazalil, thiabendazole, pyrimethanil, and fludioxonil, which are mainly employed as control agents and may have harmful effects on human health and environment. To date, numerous non-chemical postharvest treatments have been investigated for the control of these pathogens. Several studies demonstrated that biological control using microbial antagonists and natural products can be effective in controlling postharvest diseases in citrus, as well as the most used commercial fungicides. Therefore, microbial agents represent a considerably safer and low toxicity alternative to synthetic fungicides. In the present review, these biological control strategies as alternative to the chemical fungicides are summarized here and new challenges regarding the development of shelf-stable formulated biocontrol products are also discussed.

The biocontrol efficiency of Aureobasidium pullulans strain ACBL-77 against Geotrichum citri-aurantii, causal agent of sour rot in citrus, and their interactions were evaluated. For this, were evaluated the incorporation of nutrients in optimizing the antagonistic activity of the yeast, the competition for nutrients between microorganisms, the effect of nutrients on yeast cell and biofilm production and their correlation in the biocontrol of disease, the survival of yeast in citrus fruits and the interaction between microorganisms using scanning electron microscopy. Micronutrients (boric acid, cobalt chloride and ammonium molybdate) favoured the antagonistic action of A. pullulans. Ammonium sulfate 1% and sucrose 0.5% favoured the yeast during the competition between the microorganisms. The addition of ammonium sulfate (1%) in the yeast culture stimulated biofilm production and increased the antagonistic activity against the disease, as also allowed the better survival of yeast in wounded sites of citrus fruit. The yeast was found to be able to form biofilms on citrus, deforming the pathogen hyphae. These results showed the importance of the addition of nutrients in A. pullulans based-formulations when aiming for their use on a commercial scale. This is the first report of a positive correlation between the increase in the quantity of biofilm produced by A. pullulans, with increased antagonistic activity.

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.

In the agricultural sector, citrus is one of the most important fruit genus in the world. In this scenario, Brazil is the largest producer of oranges; 34% of the global production, and exporter of concentrated orange juice; 76% of the juice consumed in the planet, summing up US$ 6.5 billion to Brazilian GDP. However, the orange production has been considerable decreasing due to unfavorable weather conditions in recent years and the increasing number of pathogen infections. One of the main citrus post-harvest phytopathogen is Penicillium italicum, responsible for the blue mold disease, which is currently controlled by pesticides, such as Imazalil, Pyrimethanil, Fludioxonil, and Tiabendazole, which are toxic chemicals harmful to the environment and also to human health. In addition, P. italicum has developed considerable resistance to these chemicals as a result of widespread applications. To address this growing problem, the search for new control methods of citrus post-harvest phytopathogens is being extensively explored, resulting in promising new approaches such as biocontrol methods as “killer” yeasts, application of essential oils, and antimicrobial volatile substances. The alternative methodologies to control P. italicum are reviewed here, as well as the fungal virulence factors and infection strategies. Therefore, this review will focus on a general overview of recent research carried out regarding the phytopathological interaction of P. italicum and its citrus host.

Penicillium italicum (Blue mold) is a major postharvest disease of citrus. An alternative to controlling the disease is through the use of yeasts. The purpose of the present study was to screen effective yeast antagonists against P. italicum, isolated from soil, leaves, flowers, and citrus fruits, to assess the action mechanisms of the yeast isolates that were demonstrated to be effective for biocontrol, and to identify the most effective yeast isolates for the biocontrol of blue mold. The in vitro assays showed that six yeast strains inhibited up to 90% of the pathogen's mycelial growth. In vivo assays, evaluating the incidence of blue mold on sweet oranges, the strains ACBL-04, ACBL-05, ACBL-10 and ACBL-11 were effective, demonstrating the potential for the blue mold control when preventively applied, whereas the ACBL-08 strain showed a high potential to preventive and curative applications. Additional studies on the modes of action of these yeast strains showed that most of the evaluated yeast strains did not produce antifungal substances, in sufficient quantities to inhibit the pathogen growth. Competition for nutrients was not a biocontrol strategy used by the yeast strains. The 'killer' activity might be the main action mechanism involved in P. italicum biocontrol. This study indicated that the multiple modes of action against the pathogen presented by yeasts may explain why these strains provided P. italicum control under in vitro and in vivo conditions. However, further studies in future might be able to elucidate the 'killer' activity and its interaction with pathogen cells and the bioproduct production using Candida stellimalicola strains for control postharvest diseases.

RESUMO -A severidade da mancha-marrom de alternária nos pomares brasileiros de tangerinas tem causado sérias preocupações aos citricultores, devido aos prejuízos em plantios comerciais de variedades suscetíveis, como a tangerina Ponkan e o tangor Murcott. Para avaliar a resposta de diferentes genótipos ao fungo, foram realizadas inoculações de Alternaria alternata in vitro e in vivo, em 54 diferentes genótipos de tangerinas e seus híbridos, selecionados no Banco Ativo de Germoplasma de Citros do Centro APTA Citros Sylvio Moreira, do Instituto Agronômico, em Cordeirópolis-SP, visando a encontrar variedades resistentes. Para isso, inicialmente, testes de patogenicidade foram realizados com dez isolados de A. alternata para a seleção dos mais agressivos. Posteriormente, foram realizadas inoculações em folhas destacadas e em plântulas e, aos dois (in vitro) e três (in vivo) dias após, fez-se a contagem do número de lesões/folha e a estimativa da severidade da doença com auxílio de escala diagramática (in vivo). A maior parte dos genótipos apresentou sintomas da doença, porém com diferentes graus de suscetibilidade. Genótipos como a tangerina Sul da África e o tangelo Orlando foram os mais suscetíveis. Por outro lado, o grupo das satsumas e mexericas, assim como algumas tangerinas mostraram-se resistentes, indicando novas opções para a citricultura nacional. Termos para indexação: mancha-marrom de Alternária, Citrus reticulata, resistência varietal. RESPONSE OF DIFFERENT TANGERINE VARIETIES AND HYBRIDS TO IN VITRO AND IN VIVO INOCULATION OF Alternaria alternataABSTRACT -The confirmation of a new disease in the Brazilian tangerine orchards, the alternaria brown spot, has been concerning citrus growers because of the serious damages to commercial crops of susceptible varieties, such as Ponkan tangerine and Murcott tangor. In this research, in vitro and in vivo inoculations of Alternaria alternata fungus were held in 54 different varieties of the tangerine group selected from the Citrus Germoplasm Bank of the Citros Center APTA Sylvio Moreira, from the Instituto Agronomico, in Cordeirópolis, State of São Paulo, Brazil, aiming to find varieties with more resistance. First, a pathogenicity test with ten different A. alternata isolates was made to select the most aggressive one. After this, inoculations in detached leaves and seedlings were held and, approximately two (in vitro) and three (in vivo) days after, the number of lesions in each leaf was counted, and the disease severity estimated with diagrammatic scale help (in vivo). More than a half of varieties showed symptoms of the disease, however with different levels of susceptibility. Varieties like South African tangerine and Orlando tangelo were the most susceptible; on the other hand the satsumas and willow leaf group and some tangerines, showed resistance, indicating new options to the Brazilian citriculture.