Biocontrol of Phyllosticta citricarpa by Bacillus spp.: biological and chemical aspects of the microbial interaction.

Citrus fruits are the most produced fruits in the world, but they are threatened by several pathogens, including the fungus Phyllosticta citricarpa, the causal agent of citrus black spot (CBS). The fungus affects most citrus species and the infection results in economic losses in citrus-producing areas. This disease causes the aesthetic depreciation of fresh fruit, impairing its commercialization. As an alternative to the use of synthetic fungicides to control the pathogen, the biological control, using bacteria of the genus Bacillus, is highlighted. Such microorganisms enable biocontrol by the production of volatile organic compounds (VOC) or non-volatile. Therefore, this work aimed to evaluate the production of VOC by isolates of Bacillus spp. grown in different culture media; to evaluate the effects of these compounds on the evolution of CBS lesions in orange fruits; to study the effects of VOC on resistance induction in orange fruits; to evaluate the effects of VOC on P. citricarpa morphology in CBS lesions, and to identify the produced VOC. Tryptone soya agar (TSA) and tryptone soya broth (TSB) media used to culture the bacterium resulted in up to 73% pathogen inhibition by VOC. Volatile compounds from Bacillus spp. ACB-65 and Bacillus spp. ACB-73 when cultured in TSB culture medium provided 86% inhibition of freckles that evolved to hard spots. The volatile fractions produced by the bacteria were identified as alcohols, ketones, amines, ethers, aldehydes and carboxylic acids that can serve as arsenal against the phytopathogen. The present work demonstrated the potential of VOC produced by Bacillus spp. in the control of P. citricarpa.

Penicillium italicum: An Underexplored Postharvest Pathogen.

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.

Production of antifungal compounds by Bacillus spp. isolates and its capacity for controlling citrus black spot under field conditions.

Orange juice is a major agricultural product, and oranges are among the most widely sold fresh fruits in several countries. Citrus black spot (CBS), caused by the fungus Phyllosticta citricarpa, affects almost every species of citrus, causing a esthetic depreciation of the fruit and fruit drop, with consequent financial loss for its in natura commercialization. Fungicides are the major control measure for CBS, but have limited efficiency and high cost, and give rise to fungal strains resistant to these products. This work assayed the production of antifungal compounds by Bacillus spp. isolates and tested the potential of two isolates from B. subtilis (ACB-AP3 and ACB-83) for controlling CBS under field conditions with two previously untested orange varieties. A total of 15 isolates produced cell-free, thermostable or volatile compounds effective in suppressing P. citricarpa growth in vitro. It was possible to detect the production of two antibiotics (iturin and surfactin) by B. subtilis ACB-83. Additionally, B. subtilis isolates ACB-AP3 and ACB-83 displayed significant effects in controlling the disease under field conditions.

Biological Control of Citrus Postharvest Phytopathogens.

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.

Comparative proteomics of two citrus varieties in response to infection by the fungus Alternaria alternata.

Alternaria brown spot (ABS) is a disease caused by the necrotrophic fungus Alternaria alternata, which induces necrotic lesions on fruits and young leaves due to the production of the host-specific ACT toxin by the fungus. To better understand the citrus-A. alternata interaction and to identify putative resistance proteins, as well as the receptor of the ACT toxin, citrus plants susceptible ('Minneola' mandarin) and resistant ('Clemenules' tangor) to A. alternata, infected or not (control) with the pathogen were analyzed by proteomics. Protein changes were observed between citrus genotypes after infection, and 150 candidate proteins were obtained. A general scheme of the metabolic processes involved in susceptible and resistant citrus-A. alternata interactions was designed. Susceptible plants presented a high level of proteins involved in stress response at the final stages of the infection, whereas resistant plants presented high level of ROS proteins, metabolic proteins, and proteins involved in the immune system process. Proteins like ferredoxin and cyclophilin are specific to the susceptible variety and may be good candidates as fungal effector-interacting proteins. This is the first citrus-A. alternata proteomics analysis, which has allowed a better understanding of the molecular bases of the citrus response to ABS disease.

Antifungal activity and action mechanisms of yeasts isolates from citrus against Penicillium italicum.

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.

Biofilm production by Aureobasidium pullulans improves biocontrol against sour rot in citrus.

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.

Bacillus subtilis based-formulation for the control of postbloom fruit drop of citrus.

Postbloom fruit drop (PFD) caused by Colletotrichum acutatum affects flowers and causes early fruit drop in all commercial varieties of citrus. Biological control with the isolate ACB-69 of Bacillus subtilis has been considered as a potential method for controlling this disease. This study aimed to develop and optimize a B. subtilis based-formulation with a potential for large-scale applications and evaluate its effect on C. acutatum in vitro and in vivo. Bacillus subtilis based-formulations were developed using different carrier materials, and their ability to control PFD was evaluated. The results of the assays led to the selection of the B. subtilis based-formulation with talc + urea (0.02 %) and talc + ammonium molybdate (1 mM), which inhibited mycelial growth and germination of C. acutatum. Studies with detached citrus flowers showed that the formulations were effective in controlling the pathogen. In field conditions, talc + urea (0.02 %) provided 73 % asymptomatic citrus flowers and 56 % of the average number of effective fruit (ANEF), equating with fungicide treatment. On the contrary, non-treated trees had 8.8 % of asymptomatic citrus flowers and 0.83 % ANEF. The results suggest that B. subtilis based-formulations with talc as the carrier supplemented with a nitrogen source had a high potential for PFD control.

Bacteria as growth-promoting agents for citrus rootstocks.

The microbial community plays an essential role in maintaining the ecological balance of soils. Interactions between microorganisms and plants have a major influence on the nutrition and health of the latter, and growth-promoting rhizobacteria can be used to improve plant development through a wide range of mechanisms. Therefore, the objective of the present study was to evaluate bacteria as growth-promoting agents for citrus rootstocks. A total of 30 bacterial isolates (11 of Bacillus spp., 11 actinobacteria, and 8 lactic acid bacteria) were evaluated in vitro for indoleacetic acid production, phosphate solubilization, and nitrogen (N) fixation. In vivo testing consisted of growth promotion trials of the bacterial isolates that yielded the best results on in vitro tests with three rootstocks: Swingle citrumelo [Citrus×paradisi Macfad cv. Duncan×Poncirus trifoliata (L.) Raf.], Sunki mandarin (Citrus sunki Hort. ex Tan), and rangpur (Citrus×limonia Osbeck). The parameters of interest were height, number of leaves, stem diameter, shoot and root dry mass, and total dry mass at 150days after germination. The results showed that most bacterial isolates were capable of IAA production. Only one lactic acid bacterium isolate (BL06) solubilized phosphate, with a high solubilization index (PSI>3). In the actinobacteria group, isolates ACT01 (PSI=2.09) and ACT07 (PSI=2.01) exhibited moderate phosphate-solubilizing properties. Of the Bacillus spp. isolates, only CPMO6 and BM17 solubilized phosphate. The bacterial isolates that most fixated nitrogen were BM17, ACT11, and BL24. In the present study, some bacteria were able to promote growth of citrus rootstocks; however, this response was dependent on plant genotype and isolate. Bacillus spp. BM16 and CPMO4 were able to promote growth of Swingle citrumelo. In Sunki mandarin plants, the best treatment results were obtained with BM17 (Bacillus sp.) and ACT11 (actinobacteria). For Rangpur lime rootstock, only BM05 (Bacillus sp.) was able to promote increase in two parameters assessed, height and number of leaves. When the bacterial isolates were used in mixture there was not promoted growth of plants on rootstocks. This fact may be associated with the different mechanisms of action of each bacteria involved or with the presence of competition among the microorganisms of the mixture.

Biocontrol ability and putative mode of action of yeasts against Geotrichum citri-aurantii in citrus fruit.

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.

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.