​New challenges

Benzimidazole fungicides, a group that includes carbendazim and methyl thiophanate, have long been used to control the main fungal diseases that occur in citrus, such as floral rot, black spot, wart, melanosis and stem rot.

However, in January 2012, the USA banned the landing of ships containing Brazilian citrus juice that had carbendazim residues above the permitted level (≤ 10ppb). Therefore, in February 2012, the Brazilian industry suspended exports of concentrated and frozen orange juice to the American market.

From January to March 2012, a total of 14 shipments from Brazil, 12 from Canada and one from the Dominican Republic were rejected for the same reason, according to data from the U.S. Food & Drug Administration on March 1, XNUMX.

As a consequence of what happened, benzimidazoles were removed from the list of pesticides for integrated citrus production and farmers were left with the following question: how to control these diseases in the absence of benzimidazoles?

For the chemical industry, the path is to look for new chemical molecules that present the same control efficiency, however, when the objective is to search for alternatives for sustainable agriculture, the best path is to improve cultural and biological management measures for main fungal diseases that occur in citrus, especially for black spot, where the two reproductive phases (ascospores and pycnidiospores) of the fungus Guignardia citricarpa occur naturally in the field.

To better understand how the disease occurs and persists in the orchard, it is necessary to understand a little about its cycle. Black spot or black spot, as it is also called and known by farmers, is caused by the fungus Guignardia citricarpa, whose imperfect form corresponds to the Phyllosticta citricarpa.

G. citricarpa produces reproductive structures called pseudothecia and ascospores that are formed in decomposing leaves in the soil, constituting the primary source of the fungus inoculum, responsible for the best-known symptoms of the disease, black spot. The beginning of these reproductive structures of the fungus (ascospores) occurs approximately 50 to 180 days after the leaves fall and normally the discharge of ascospores follows the rain, in such a way that three months may be enough for their release, as long as there is previous moisture on the leaves. Wetting and drying the leaves alternately are vital for the development of these structures.

On the surface of susceptible organs, ascospores germinate and produce fixation and penetration structures, reaching the cuticle, forming a small mass of quiescent subcuticular mycelium. The fungus can remain dormant for up to 12 months. This period of dormancy can be interrupted when the citrus fruit reaches its final size and begins to mature or when environmental conditions become favorable. The fungus then grows from the subcuticular mycelium and colonizes the innermost tissues, which results in the appearance of the typical symptoms of the disease.

The natural fall of citrus plant leaves can occur, to a greater or lesser extent, throughout the year. However, some factors such as water stress, nutritional imbalance, pests and diseases can aggravate this abscission. Because ascospores are formed on these leaves, there is theoretically the potential for these structures to be formed year-round. However, the prevailing climatic conditions have a greater or lesser impact on its viability and speed of production.

The imperfect phase of the fungus (P. citricarpa) produces reproductive structures called pycnidiospores, which are formed in fruit lesions and leaves and are easily carried by water to the surface of susceptible organs located below. Therefore, these structures are responsible for the spread of the fungus over short distances. Pycnidiospores are important in disease epidemics when infected fruits containing these spores and young susceptible fruits coexist on the same plant.

Given this knowledge, it becomes easier to understand the strategies that should be adopted to control the disease, including cultural and biological measures.

Cultural measures to control black spot include using pathogen-free citrus seedlings; care with the harvest, avoiding the movement of vehicles within the orchard that could carry contaminated leaves, in addition to being careful not to transport and sell fruits with leaves; remove fallen leaves on the soil surface, if possible; prune and remove dry branches, considering that the fungus (G. citricarpa) survives in this type of material; use windbreaks within the orchard, which prevents the spread of reproductive structures (ascospores) of the fungus to other areas; take care of irrigation, so that flowering is standardized and leaf fall is reduced during the dry period of the year, since ascospores are formed in leaves that fall to the ground and their production is stimulated by alternating wetting and drying of the leaves. sheets; maintain plants in good nutritional and health conditions; provide mulch over the leaves that have fallen to the ground and, to this end, we suggest managing the weeds using an ecological chair, and, finally, bringing forward the harvest.

Biological control measures refer to techniques that are adopted in order to increase and sustain the biological interactions on which agricultural production is based, rather than simply reducing or simplifying them. Considering the etiology and epidemiology of G. citricarpa, it is assumed that measures that may culminate in accelerating the decomposition of citrus leaves fallen under the canopy may inhibit the formation of pseudothecia and, consequently, reduce the source of inoculum represented by ascospores.

According to the literature, there are several compounds that have the property of accelerating the decomposition of plant material. These compounds include nitrogen fertilizers, which can promote the activity of microorganisms with cellulolytic activity or even the application of commercial products formulated for this purpose. It is also possible to use microorganisms with the potential to act directly on the decomposition of plant material or that have direct action on the pathogen present in these leaves that fall to the ground, as in the case of G. Citricarpa, contributing to reducing the severity of the disease.

Experiments carried out at the Sylvio Moreira Citriculture Center, in Cordeirópolis (SP), one of the units of the Campinas Agronomic Institute (IAC), sought to investigate the use of biological control agents, two isolates of Trichoderma (ACB-37 and ACB-40) and two isolates from Bacillus subtilis (ACB-83 and ACB-AP3), during disease management under field conditions. To this end, microorganisms were applied to citrus leaves that had fallen to the ground, to reduce the production and viability of ascospores by accelerating their decomposition, as an auxiliary control measure. The results showed that the lowest disease rates were obtained with applications of the bacteria isolates.

At the same time, the control efficiency of isolates of Bacillus was tested on Valencia orange and Pera orange plants, belonging to commercial orchards in the municipality of Araras/São Paulo. Products based on microorganisms were applied to the plants every 28 days, in two concentrations (05% and 10%), in the presence or absence of mineral oil and compared with the standard chemical treatment. The results showed the potential of the ACB-AP3 isolate (05%) to control the disease, regardless of the presence or absence of mineral oil during treatment.

The good performance of the bacterial isolates shown in the experiments may be due mainly to their mechanism of action. Reports in the literature show that antibiosis is the main mechanism involved in antagonistic activity, both through the production of antibiotics and the production of aminolytic and proteolytic enzymes.. These compounds can lyse and dissolve cellular structures regardless of contact. In addition to its mechanism of action, this microorganism has ready adaptability in the environment, mainly because it is a spore-forming bacterium, which offers tolerance to different types of stress. such as drought, nutritional deficiencies and extreme temperatures. These characteristics, added to the easy development and lack of pathogenicity of B. subtilis, suggest stable and viable product formulations for controlling phytopathogens. To this end, there is a need to continue testing, studying the application times, concentration and appropriate formulation of antagonists for better control of black spot.

Click here to read the article in Cultivar Vegetables and Fruits 82.