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Revista mexicana de fitopatología
versión On-line ISSN 2007-8080versión impresa ISSN 0185-3309
Rev. mex. fitopatol vol.36 no.1 Texcoco ene./abr. 2018
https://doi.org/10.18781/r.mex.fit.1708-1
Phytopathological notes
Selection in vitro of mycoparasites with potential for biological control on Coffee Leaf Rust (Hemileia vastatrix)
1 Centro Regional Universitario Oriente. Universidad Autónoma Chapingo, Carretera Huatusco-Xalapa, Km 6, CP. 94100, Huatusco, Veracruz.
2 Campo Experimental Rosario Izapa. INIFAP. Carretera Tapachula-Cacahoatan, Km 18, CP. 30870, Tuxtla Chico, Chiapas.
3 Instituto de Ecología, A. C. Carretera antigua a Coatepec No. 351, El Haya, CP. 91070, Xalapa, Veracruz.
4 Instituto Tecnológico Superior de Xalapa, Sección 5ª de la Reserva Territorial S/N, CP. 91060, Xalapa, Veracruz.
In order to isolate and identify mycoparasites of Hemileia vastatrix and to know their potential as a biological control for Coffee Leaf Rust, from December 2014 to January 2015, samples of Arabica coffee with pustules and the presence of possible mycoparasites were sampled. The fungi associated with the pustules were isolated and identified morphometrically at the genus level. The percentage of mycoparasitism in vitro of three of the isolates on rust pustules was evaluated. We obtained 23 isolates of microorganisms associated with rust pustules: Lecanicillium spp. (7), Calcarisporium sp. (4), Sporothrix sp. (4) and Simplicillium spp. (8). All the isolates evaluated showed mycoparasitism in rust uredospores; however, 120 h after inoculation, the highest percentages (P = 0.05) were obtained with Simplicillium sp. (89%) and Lecanicillium sp. (68%).
Key words: Lecanicillium; Calcarisporium; Sporothrix; Simplicillium
Con el objetivo de aislar e identificar micoparásitos de pústulas de Hemileia vastatrix y conocer su potencial de control de la roya de café, de diciembre del 2014 a enero del 2015 se muestrearon hojas de café arábico con pústulas de roya y presencia de posibles micoparásitos. Los hongos asociados a las pústulas se aislaron e identificaron morfométricamente a nivel de género. Se evaluó el porcentaje de micoparasitismo in vitro de tres de los aislamientos sobre pústulas de roya. Se obtuvieron 23 aislamientos de microorganismos asociados a pústulas de roya: Lecanicillium spp. (7), Calcarisporium sp. (4), Sporothrix sp. (4) y Simplicillium spp. (8). Todos los aislamientos evaluados mostraron micoparasitismo en las uredosporas de roya; sin embargo, 120 h después de la inoculación, los mayores porcentajes (P=0.05) se obtuvieron con Simplicillium sp. (89%) y Lecanicillium sp. (68%).
Palabras clave: Lecanicillium; Calcarisporium; Sporothrix; Simplicillium
Coffee Leaf Rust is caused by the fungus Hemileia vastatrix, a biotrophic pathogen that affects Coffea leaves, and is considered the most important disease on this crop worldwide. This fungus causes defoliation and reduces the yields of the coffee plants (Avelino et al., 2015). The severity of the recent rust epidemics in Central America and Mexico has caused losses of 40 to 50% in the yield of the crop (Cressey, 2013). According to the International Coffee Organization (ICO, 2015), economic factors (decapitalization of farmers) and agronomic factors (lack of management of the crop) have contributed to reach these losses.
In response to the coffee crisis caused by Coffee Leaf Rust, short-term actions were implemented in Central America; however, the main strategies for their management focus particularly on chemical control and the use of resistant varieties (Zambolim et al., 1997; Avelino et al., 2015 and Escamilla, 2016).
Chemical control is considered scarcely promissory for Mexican coffee growing activities, since it is a socially important crop, as well as considering the organic farming sector. Biological control is an alternative, boosting the use of microorganisms that come from the same action environment as the plant pathogen.
Within the strategies for the biological control of plant pathogens, one of the options is the use of mycoparasites, fungi that are able to survive at the expense of another fungus (Boosalis, 1964), affecting the reproductive structures of the pathogen, which limits its development and spreading (Barros et al., 1999).
Different mycoparasites have been reported on plant pathogens, including Trichoderma sp. and Penicillium vermiculatum on Rhizoctonia solanii (Rolz et al., 2013); Calcarisporium parasiticum, as well as Physalospora spp., and Trichoderma spp. only Armilaria mellea (Boosalis, 1964) and other fungi such as Sclerotinia spp. (Hoyos et al., 2008), Rhizoctonia solani, Phytophthora nicotianae, P. capsici and Pythium aphanidermatum, on Penicillium sp. and Fusarium sp. (Sandoval and López, 2001). Within the group of rusts, the main mycoparasitic relations reported are: Cladosporium tenuissimum on Uromyces apendiculatus (Assante et al., 2004); Cladosporium uredinicola on Puccinia puta (Barros et al., 1999); Simplicillium lanosoniveum affecting Phakopsora pachyrizi (Gauthier et al., 2014) y Verticillium lecanii en Puccinia recóndita (Spencer and Atkey, 1981).
Given the importance of the Coffee Leaf Rust disease, different Studies have been carried out to determine the species with mycoparasitism related to H. vastatrix (Carrión, 1988; Carrión and Rico, 2002; Mahfud et al., 2006; Rolz, 2013 and Haddad et al., 2014); however, most of them only recorded the presence of mycoparasites of Coffee Leaf Rust and not their potential as possible agents of biological control of the disease.
Based on these precedents, the aim of this investigation is to isolate and identify mycoparasites of H. vastatrix pustules, as well as to evaluate their potential to control the disease in vitro.
Materials and methods
Collection sites. In an altitudinal profile in the state of Veracruz, three coffee-growing locations were chosen: Matlaluca, in the municipal area of Zentla (at an altitude of 650 masl, an average annual temperature of 22°C and 1300 mm of yearly rainfall); El Ocote (at an altitude of 1030 masl, an average annual temperature of 19.8°C and 1682 mm of yearly rainfall) and Tlavictepan (1250 masl, an average annual temperature of 17.2°C and 1967 mm of yearly rainfall), in the municipal area of (Table 1).
Table 1 Description of the sites of collection of mycoparasites of the Coffee Leaf Rust.
| Localidad | Coordenadas | Altitud (msnm) |
Temperatura media anual (°C) |
Precipitación media anual (mm) |
Sistema de producción |
Variedad |
| Matlaluca | 19°07´56´´ 96°46´30´´ |
650 | 22 | 1300 | Policultivo Tradicional |
Typica, Bourbon |
| El Ocote | 19°07´56´´ 96°53´30´´ |
1030 | 19.8 | 1682 | Policultivo comercial |
Costa Rica, Colombia, Typica |
| Tlavictepan | 19°09´40´´ 96°56´34´´ |
1250 | 17.2 | 1967 | Policultivo comercial |
Costa
Rica, Colombia, Typica |
Note. According to the classification by Nolasco and Toledo (1996), a commercial polyculture system is one which used different combinations of introduced forest and fruit trees, including weed control and the selective trimming of coffee plants and without phytosanitary management, whereas commercial polyculture uses commercial shade species, carrying out general and particular tasks in the crops.
Sampling of plant material. In each location, five plots, each with a surface area of one hectare, samples were taken of plants with symptoms of rust and with signs of possible mycoparasites in the pustules. The collection period was between December 2014 and January 2015, and in each plot, 40 leaves were taken from different coffee plants. The leaves were wrapped in sterile paper towels and placed inside properly labeled plastic bags. They were transferred to the Fungus Biodiversity and Systematics of the Environment Institute, A. C. Lab for their preparation and analysis.
Isolation of mycoparasites. In the lab, six rust pustules were taken from each leaf, with signs of possible mycoparasites. Under a stereoscopic microscope (Leica® Heerbrug, Suiza), and with the aid of a hypodermic needle, portions of fungal mycelia related to rust pustules were taken and placed in Petri dishes with oat agar culture medium with an antibiotic (Cloranfenicol® Toluca, México) at 1%. The cultures were kept at 24+1°C for ten days and purified by hyphal tip isolation.
Taxonomic identification. The morphometric characterization of the pure isolations was carried out with the production of temporary and permanent preparations of the isolations, followed by their observation under a compound microscope (Leica, DM550®, Heerbrug, Switzerland) and the software Leica Aplication Suite Educational Zoom (LAS EZ) version 3.0. The genus identification considered the presence and dimensions of phialides and conidia (n=30), and they were described with the aid of specialized taxonomic keys: Barranco (2004), Barnet and Lilly (1958), Domsch et al., (1980), Hirose et al., (2011), Hoog (1974), Zare et al. (2000), Zare and Gams, 2001.
Evaluation of the mycoparasitic potential of isolated microorganisms. Three isolations (Lecanicillium sp.-TlaP4, Simplicillium sp.-MaP2 and Calcarisporium sp.-OcP2) were selected for the evaluation of their potential as H. vastatrix mycoparasitic microorganisms; the criterion for selection was the growth rate of the isolations. The isolations were incubated in Potato Dextrose Agar (PDA) for 10 days, followed by the production of a spore suspension at a concentration of 5x106 spores ml-1; each suspension was added an aliquot of Tween 20® at 0.01%.
The test in vitro to evaluate the mycoparasitism was carried out according to the methodology proposed by Eskes et al. (1991), with adaptations. Leaves of Coffea arabica plants of the bourbon variety, with symptoms of Coffee Leaf Rust, were taken in the field and kept in polypaper bags for their analysis in the lab. Under a stereoscopic microscope, pustules were analyzed to select only those that presented no mycoparasitic microorganisms. Five leaf disks, each 1.5 cm in diameter, with the presence of at least one rust pustule, were placed in a Petri dish with agar water; each leaf disk was considered as a repetition of five. We sprayed 1 ml of the spore suspension previously prepared with each one of the strains, and the control group was sprayed with sterile distilled water. The treatments were established under a totally random design and kept under light periods of 12 h and 12 h of darkness, at 24 °C.
To determine the percentage of mycoparasitism in rust pustules, in each leaf disk, the sporulating lesions were scraped and suspended in 1 ml of distilled water, adding 100 µl of Tween 80 at 0.01%. From the dilution obtained, 100 µl were taken and observed under the microscope to count the number of parasited and non-parasited uredospores in the sample. The evaluations were carried out every 24 hours for a period of 5 days.
Statistical analysis. The percentage of parasitism in the uredospores evaluated every 24 hours was analyzed with an analysis of variance and a Tukey average separation test, with the aid of the program Statistica version 6.0.
Results and discussion
Isolation of mycoparasites. A total of 23 mycoparasite isolations were obtained from the different sampling sites. The highest number (10) was obtained from the middle altitude zone (El Ocote, 1030 masl), and the lowest number (6), from the low altitude zone with a higher temperature and rainfall than the other sampling sites (Zentla, 650 masl). Considering that weather conditions are factors that determine the development of fungi, our results differ from those reported by Martins et al., (2015), who report a greater incidence of mycoparasites on H. vastatrix in the dry season of the year, and also suggest that the mycoparasite Lecanicillium lecanii could be more persistent at lower temperatures. This is also different to reports by Bagyaraj et al., (2015) who found a greater bacteria and fungus populations in rustican systems, and therefore, in this case, there are reports of a higher diversity in conditions of commercial polyculture, where shade is less diverse than in the lower zone site.
Four fungal genera were identified: Calcarisporium sp. (4), Lecanicillium spp. (7), Simplicillium spp. (8), Sporothrix sp. (4), with Simplicillium spp. and Lecanicillium spp. being the most abundant.
Microscopic description of the genera.
Lecanicillium spp. Presented septated hyphae, hyalines; with con phialides (15-23 x 0.5-1.2 µm) ordered in groups of three to five per whorl (Zare and Gams, 2001), and not in pairs or individually, as pointed out by Cañedo and Ames (2004); they were also slightly wider at the base. The conidia were elliptical (5-7x1-2 µm) and emerging in the top of the phialide (Figure 1 A); they were generally observed in conidial heads (Barranco, 2004).
Figure 1 Mycoparasites found in Coffee Leaf Rust pustules (Hemileia vastatrix) and their morphological characteristics in Oat-Agar. Where: Lecanicillium sp. (A-40X); Calcarisporium sp. (B-40X); Simplicillium sp. (C-40X) and Sporothrix sp. (D-40X). Bar: 10 µm.
Calcarisporium sp. Presented hyaline and septated hyphae, which hold short conidiphora (Figure 1 B), and on them, verticillated phialides measuring 4-6 x 1-2 µm. A characteristic that distinguishes this genus is the shape of the phialides, which appeared wider in the base and end in small denticles on the apex (Hirose et al., 2011). The simple phialides are formed directly from the hypha (Barnet, 1958). The conidia are ovoidal and measure 3-5x1.3-1.8 µm.
Simplicilllium spp. Presented hialine, this and septated hyphae; the conidiophora (15-35x0.8-1µm) emerge in ones (Zare et al., 2000), perpendicular to the hyphae and become thinner towards the tip (Molina et al., 2012). Hyphae are generally interlaced (Figure 1 C). The conidia (1-3 µm) were circular and sometimes elliptical, with an abundant sporulation and an arrangement in mucoidal heads (Domsch et al., 1980).
Sporothrix sp. Presented hyalin vegetative hyphae, with short septa and denticles (Figure 1 D); no conidiophora were observed after 10 days; the conidia were observed on the denticles of the hyphae (Hoog, 1974); there was an abundant sporulation in the Agar-Oat medium.
Mycoparasitism in vitro on H. vastatrix. Forty-eight hours after the inoculation of treatments, significant statistical differences (P=0.05) were obtained between treatments (Table 2). The highest percentages of mycoparasitism observed in the first 72 hours were found in isolations of Simplicillium sp., and Calcarisporium sp., whereas 96 and 120 h after inoculation, the highest percentages were found in Simplicillium sp. and Lecanicillium sp. (Table 2).
Table 2 Evaluation of the percentage of parasitismo of three microorganisms on Coffee Leaf Rust pustules (Hemileia vastatrix) in in vitro conditions.
| Tratamiento | Horas después de la inoculación | ||||
| 24 | 48 | 72 | 96 | 120 | |
| Testigo | 0.00+0a | 0.00+b | 0.00+c | 0.00+c | 0.00+0c |
| Calcarisporium sp. | 0.00+0a | 20.87+13ab | 41.02+13ab | 50.12+0.7b | 51.60+10b |
| Lecanicillium sp. | 2.68+2 a | 9.85+6b | 17.49+16bc | 49.19+13b | 68.10+12b |
| Simplicillium sp. | 0.89+2 a | 42.73+9az | 51.19+16az | 83.48+3az | 88.86+11az |
z=Means +Standard deviation, values with the same letter in the columns do not differ statistically (Tukey p=0.05)
The genus Simplicillium is reported as part of the fungi related to Coffee Leaf Rust in Puerto Rico and Mexico (James et al., 2016). The species of this genus have been registered mainly in relation with H. vastatrix (Zare and Gams, 2001), while its potential as a biological control agent of other diseases has had little studies. It has been recorded in association with pathogenic fungi in plants, such as Alternaria brassicicola, Sclerotium rolfsii and Rhizoctonia solani (Shyang et al., 2017), as well as with nematodes (Gams and Zare, 2003) and aphids (Shyang et al., 2017).
The genus Lecanicillium has been widely studied, and documented as a biological control agent for mildews and uredinales (Alavo, 2015). Mahfund et al. (2006) point out that the effects of two species of this genus can vary between the decoloring of uredospores, the formation of white mycellia on them, or necrosis, depending on the time of application. This suggests that the percentage of parasitism of Lecanicillium sp. varies between species and isolations (Arriola et al., 1998).
Finally, previous studies have pointed out the natural parasitism of Calcarisporium sp. on Coffee Leaf Rust (Carrión and Rico, 2002).
Conclusions
The genera Lecanicillium sp., Calcarisporium sp., Sporothrix sp. and Simplicillium sp. were isolated from rust pustules in the municipal areas of Huatusco and Zentla, in the estate of Veracruz, Mexico.
The highest percentages of mycoparasitism in the in vitro tests were obtained with the strains of Simplicillium sp. (88.86%) and Lecanicillium sp. (68.10%), 120 hours after inoculation.
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Received: August 02, 2017; Accepted: December 11, 2017
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