Introduction

Coffee production (Coffea arabica L.) has a high economic, social and environmental value in the countries where it is grown (^{Flores, 2015}). However, the crop is affected by the attack of pests and diseases, aggravating due to climatic conditions (^{Waller, 1982}; ^{SARH, 1993}). The affectation goes from the germination of the plant to the productive stage, which generates large losses in production and a decrease in the quality of the grain (^{CEPAL and CAC/SICA, 2014}; ^{Chemura et al., 2017}).

Rust (Hemileia vastatrix Berk. & Br.) is the most important coffee disease (^{Haddad et al., 2014}; ^{Oliveira, et al., 2014}; ^{Barka et al., 2017}), when using this crop as its main host (^{Brown and Hovmøller 2002}). It generally causes the loss of leaves of up to 50% (^{Avelino et al., 2004}; ^{Bonilla, 2018}) and 30% of the yield in some varieties of C. arabica L., which means a great economic impact worldwide (^{Oliveira, et al., 2014}; ^{Barka et al., 2017}). In the 2013-2014 cycle, production in Central America and Mexico was affected by 3.3 million bags (^{Flores, 2015}), so in 2016 Mexico implemented the Phytosanitary Epidemiological Surveillance Program in the states of Hidalgo, Jalisco, State of Mexico, Nayarit, Querétaro, Chiapas, Veracruz, Puebla, Guerrero, San Luis Potosí and Oaxaca to determine the incidence, severity and implement preventive management actions against rust (^{SENASICA, 2016}).

H. vastatrix is controlled through the use of agrochemicals, fungicides, biofungicides, resistant varieties, biological control, cultural control and integrated management (^{Obando et al., 2013}; ^{Hernandez-Martinez and Velazquez-Premio, 2016}). Agrochemicals generate pollution to the environment and increase production costs (^{Romero, 2010}; ^{Gonza et al., 2013}), therefore, viable alternatives have been sought that guarantee sustainability in agricultural production (^{Gonza et al., 2013}).

An example of this is the use of cupric fungicides (copper oxychloride and mineral broths) considered the most efficient for the control of rust, since they do not alter the biota of the agroecosystem (^{Capucho et al., 2013}; ^{Melchor et al., 2018}), these penetrate the leaf tissue and have curative effects (^{McCook, 2009}) attack the fungus during mycelial growth and pustule formation (^{Duicela and Ponce, 2015}) as well as the use of microorganisms such as fungi and bacteria (^{Gómez-De La Cruz et al., 2017}), with the ability to survive at the expense of the fungus (^{Boosalis, 1964}), affecting the reproductive structures of the pathogen (^{Barros et al., 1999}).

The incidence and severity of rust is obtained through visual evaluations by trained personnel (^{Zambolim, 2016}). However, the use of logarithmic scales with illustrations of leaves in different degrees of damage is common, being a method to quantify visual severity (^{Nascimento et al., 2005}; ^{Hernández and Sandoval, 2015}). Digitized severity is obtained with a leaf area integrator (Chavarra et al., 2015) and the use of software such as ImageJ, Image Tool and Adobe Photoshop, allowing image areas to be determined with greater accuracy and precision (^{Sussel et al., 2009}; ^{Rincón et al., 2012}).

Based on the above, the objective of the present study was to evaluate the incidence and severity of rust (Hemileia vastatrix Berk. & Br.) in coffee seedlings (Coffea arabica L.) var. Geisha under the effect of different biofungicides.

Materials and methods

The study was carried out from February to August 2018 at the El Nueve farm, Santa María Huatulco, Oaxaca, located at coordinates 15° 55’ 56’’ north latitude and 96° 17’ 08’’ west longitude, with an altitude range of 1 200 to 1 300 m. The type of vegetation is median sub-evergreen forest (^{INEGI, 2019}). To know the fertility level of the substrate in the nursery (mixture of crop residues and forest soil ratio 1:1), a physical-chemical analysis was carried out according to NOM-021-RECNAT-2000 (^{SEMARNAT, 2002}).

Three-month-old var. Geisha coffee seedlings were used, transplanted into 13 x 20 cm polyethylene bags, standardized in terms of height (10 cm) and size. 24 treatments with 10 repetitions were established, each repetition was considered a seedling. The products evaluated were: a single effect control, four based on microorganisms, four based on minerals, one homeopathic and the combinations with the same dose (Bss+Nr, Bs+Nr, M+Nr, Bfg+Nr, Oc+Nr, Cb+Nr, Cmv+Nr, S+Nr, Bss+Bfg, Bs+Bsg, M+Bfg, Bss+Nr+Bfg, Bs+Nr+Bfg and M+Nr+Bfg) (Table 1).

The treatments were applied at two times one month apart (February 15 and March 15). The applications were made in the morning and the dosages were adjusted to 10% of what is applied in the field based on the recommendations issued by the manufacturers and specified on the label of the products.

The incidence of rust (INR) was evaluated using the methodology of ^{Samayoa-Juarez and Sanchez-Garita (2000}), adapted to the research work that consisted of counting the total of diseased leaves for each repetition and dividing by the total number of leaves, multiplying the result by 100. The following variables were considered for each repetition: total leaves (HT), healthy leaves (HS) and leaves damaged by rust (HDR). Five evaluations were made every 30 days after applying the biofungicides (dda). At 150 dda, the visual severity (SVR) of the HDR was determined, using the logarithmic-diagrammatic scale proposed by ^{SENASICA (2016)} (Figure 1).

Figure 1 Logarithmic-diagrammatic scale to assess the severity of leaf rust. 

At the same time, HDR photographs were taken to determine the digitized severity (SDR), considering the total leaf area (AFT), rust damaged area (ADR) and the healthy area (AS), analyzing the photographs with Adobe Photoshop^® software CC 2017. For this, 1 cm of the image was measured by standardizing the measurement scale (370 pixels= 1 cm), for all images. A completely randomized design was used. The assumptions of normality and homogeneity were applied to the data. As the test was not completed, even after the data had been transformed, they were analyzed using nonparametric statistics using the Kruskal-Wallis test using statistical software SAS/STAT 9.3 (^{SAS Institute Inc., 2011)}.

Results and discussion

The results of the analysis indicate that the texture of the substrate is loamy sand (Ac), pH of 6.9, electrical conductivity of 0.23 dS m^-1 (negligible effects of salinity) organic matter of 5.82% (high levels), phosphorus of 20.86 mg kg^-1 (average levels), calcium of 0.0011 cmol kg soil (low), magnesium of 0.0016 cmol kg soil and nitrogen of 0.38%.

The percentage of leaves with rust (PHR) showed significant differences (0.041) between biofungicides up to 150 dda, with a value of p≤ 0.05. However, from 30 to 120 dda there was no response from biofungicides on the incidence of H. vastatrix Berk. & Br. in INR microorganism-based biofungicides were more effective. Combinations stand out Bacit-Sur + Nat-Rx; Baci-Sur subtilis + Blite Free F-07/Guanobras and Michoderma + Blite Free F-07/Guanobras, which generated the greatest control over the INR of H. vastatrix with average ranges of 93.5 (Figure 2).

Figure 2 Biofungicide response to the incidence of H. vastatrix Berk. & Br. in seedlings of C. arabica L. var. Geisha at 150 dda. Low values of medium ranges equal a lower incidence. 

According to ^{Cacefo et al. (2016}) when applying B. subtilis in the var. Icatu and Mundo Novo obtained a control of 24 and 17%, respectively. Likewise, ^{Dorighello (2013}) when using isolates of B. subtilis QST 713 and AP-3 in the control of soybean rust (Phakopsora pachyrhizi), observed a reduction in the germination of uredospores. ^{Li et al. (2013)} obtained a significant reduction in the incidence of Puccinia striiformis f. sp. tritici (Pst) (soybean rust) with the application of B. subtilis E1R-j under greenhouse conditions with two different formulations at 24 and 0 h before Pst inoculation.

^{Leonel and Barros (2013}) obtained a lower incidence of H. vastatrix using a homeopathic complex, observing higher vegetative vigor. Likewise, ^{Rissato et al. (2016}) obtained the lowest incidence of white bean mold (Sclerotinia sclerotiorum) with homeopathic medicine applications. For their part, ^{El-Sharkawy et al. (2015}) used Streptomyces viridosporus in combination with Trichoderma harzianum for the control of greenhouse rust of wheat (Puccinia triticina), with which they obtained a decrease in the number of pustules per unit area (cm²).

For the SVR and SDR there were no significant differences; however, it was numerically observed that the combinations of Bs + Nr; Bss + Bfg and M + Bfg generated greater control over the severity in both variables (Figures 3a and 3b). ^{Cisneros-Rojas et al. (2017}) reported greater growth and development of coffee seedlings applying B. subtilis with individual effect and in combination with the Kocuria sp., ^{Cacefo et al. (2016}) point out that B. subtilis stimulates the plant to activate its defense mechanisms for greater resistance, mitigating the harmful effects of pathogens. According to ^{Pérez et al. (2015}) the biocontroller effect of Trichoderma spp. it has a mode of action that regulates the development of phytopathogenic fungi.

Figure 3a Response of biofungicides on the visual severity of H. vastatrix in seedlings of C. arabica L. var. Geisha, at 150 dda. Low values media ranges equivalent to less severe. 

Figure 4 Response of biofungicides on digitized severity of H. vastatrix in seedlings of C. arabica L. var. Geisha, at 150 dda. Low values media ranges equivalent to less severe. 

^{Infante et al. (2009}) indicate that, during the infection process, Trichoderma spp., deactivates the enzymes of the phytopathogenic fungi. ^{El-Sharkawy et al. (2015}) found that Trichoderma harzianum and Streptomyces viridosporus had the best effects for controlling the severity of wheat leaf rust, compared to chemical fungicides. ^{Evangelista-Martínez et al. (2015}) mention that bacteria of the genus Streptomyces are producers of secondary metabolites and enzymes that act in the control of diseases caused by fungi in plants.

Conclusions

The logarithmic-diagrammatic scale facilitated the measurement of the severity in leaves in the field; however, although this activity requires time to evaluate each of the leaves with rust, since a thorough revision of the leaves is required when very small lesions appear and resemble the images proposed on the scale. The bacterium B. subtilis is used for the biological control of coffee rust, but its effects also influence the growth and development of seedlings. B. subtilis in combination with Streptomyces sp. and bat guano, the latter rich in nutrients that help strengthen seedlings.

Which is also reflected when applying the Trichoderma harzianum fungus that helps the seedling to inhibit rust and to capture nutrients found in the soil. B. subtilis has proven to be an important option in biological control, especially of foliar diseases. Bacteria belonging to the genus Bacillus have been used in the biological control of phytopathogenic fungi. The use of B. subtilis bacteria is an alternative for the biological control of coffee rust, and it has also been used for the development of successful coffee seedlings.