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Revista mexicana de fitopatología

On-line version ISSN 2007-8080Print version ISSN 0185-3309

Rev. mex. fitopatol vol.36 n.1 Texcoco Jan./Apr. 2018

http://dx.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)

Irene Gómez-De La Cruz1 

Emiliano Pérez-Portilla1 

Esteban Escamilla-Prado1 

Misael Martínez-Bolaños2  * 

Gloria Luz L. Carrión-Villarnovo3 

Tania I. Hernández-Leal4 

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.

Abstract:

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

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.

Source: Servicio Meteorológico Nacional, 2000.

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.

Literatura citada

Alavo BC. 2015. The insect pathogenic fungus Verticillium lecanii (Zimm.) Viegas and its use for pests control: A review. Journal of Experimental Biology and Agricultural Sciences 3:337-345. http://dx.doi.org/10.18006/2015.3(4).337.345 [ Links ]

Arriola MC, Chet I and Rölz C. 1998. Hongos que atacan la roya del café: Un breve comentario. Universidad Del Valle de Guatemala 8:2-6. Disponible en línea: http://uvg.edu.gt/publicaciones/revista/volumenes/revista8.pdfLinks ]

Assante G, Maffi D, Sarachi M, Farina G, Morica S and Ragazzi A. 2004. Histological studies on the mycoparasitism of Cladosporium tenuissimum on urediniospores of Uromyces appendiculatus. Mycology 108:170-182. https://doi.org/10.1017/S0953756203008852 [ Links ]

Avelino J, Cristancho M, Georgiou S, Imbach P, Aguilar L, Bornemann G, Läderac P, Anzueto F, Hruska AJ and Morales C. 2015. The coffee rust crisis in Colombia and Central America (2008-2013): impacts, plausible causes and proposed solutions. Food Security 7: 303-321. https://doi.org/10.1007/s12571-015-0446-9 [ Links ]

Barnett HL and Lilly VG. 1958. Parasitism of Calcarisporium parasiticum on species of Physalospora and related fungi. Boletin 420T. West Virginia University Agricultural Experiment Station. 1-37 pp. Disponible en línea: https://ia902300.us.archive.org/13/items/parasitismofcalc420barn/parasitismofcalc420barn_bw.pdfLinks ]

Barranco, FJE. 2004. Contribución al estudio de las actividades enzimáticas involucradas en el mecanismo de patogenicidad de Lecanicillium (Verticillium) lecanii cultivado en medio sólido. Universidad Autónoma Metropolitana. Iztapalapa, México, D.F. [ Links ]

Barros ST, Oliveira TN, Bastos T G and Maia CL.1999. Hyperparasitism of Cladosporium uredinicola over Puccinia puta on the host Ipomoea fistulosa. Mycologist 13:23-24. https://doi.org/10.1016/S0269-915X(99)80071-8 [ Links ]

Bagyaraj JD, Thilagar J, Ravisha C, Kashalapa GC, Krishna-murthy NK and Vaast P. 2015. Below ground microbial diversity as influenced by coffee agroforestry systems in the Western Ghats, India. Agriculture, Ecosystems and Environment 202:198-202. https://doi.org/10.1016/j.agee.2015.01.015 [ Links ]

Boosalis MG. 1964. Hyperparasitism. Annual Review of Phytopathology 2:363-376. https://doi.org/10.1146/annu-rev.py.02.090164.002051 [ Links ]

Carrión G and Rico-Gray V. 2002. Mycoparasites on the coffee rust in Mexico. Fungal Diversity 11:49-60. Disponible en línea: http://www.fungaldiversity.org/fdp/sfdp/FD_11_49-60.pdfLinks ]

Carrión G. 1988. Estudios sobre el control biológico de la roya del cafeto mediante Verticillium lecanii en México. Micología Neotropical 1:79-86. [ Links ]

Cressey D. 2013. Coffee rust regains foothold: researchers marshal technology in bid to thwart fungal outbreak in Central America. Nature 493 (7434):587. Disponible en línea: http://www2.tap-ecosur.edu.mx/mip/Plagas/Roya/pdfs/Notas/Coffee%20rust%20regains.pdfLinks ]

Domsch KH, Gams W and Anderson TH. 1980. Compendium of soil fungi. Vol.1. Academic Press. London, UK. 860p. [ Links ]

Escamilla PE. 2016. Boletín informativo: Políticas públicas. Las variedades de Café en México ante el desafío de la Roya. Inédito. Disponible en línea: http://pmcarbono.org/pmc/descargas/proyectos/redd/Breves_de_Politicas_Publicas_No.4-Variedades_de_cafe_en_Mexico.pdfLinks ]

Eskes AB, Mendes MD and Robbs CF. 1991. Laboratory and field studies on parasitism of Hemileia vastatrix with Verticillium lecanii and V. leptobactrum. Café-Cacao-Thé, 35:275-282. [ Links ]

Gauthuier WN, Maruthachalam K, Subbarao VK, Browm M, Xiao Y, Robertson L and Schneider WR. 2014. Mycoparasitism of Phakosphora pachyrizi, the soybean rust pathogen, by Simplicillium lanosoniveum. Biological Control 76:87-94. http://dx.doi.org/10.1016/j.biocontrol.2004.05.008 [ Links ]

Gams W and Zare R. 2003. A taxonomic review of the clavicipitaceous anamorphs parasitizing nematodes and other microinvertebrates. 17-73. In: Clavicipitalean Fungi: Evolutionary biology, Chemistry, Biocontrol and Cultural Impacts. DOI: 10.1201/9780203912706.pt1 [ Links ]

Haddad, F, Saraiva R, Mizubuti E, Romeiro R and Maffia L. 2014. Isolation and selection of Hemileia vastatrix antagonists. European Journal of Plant Pathology 139:763-772. https://link.springer.com/article/10.1007/s10658-014-0430-9Links ]

Hirose D, Dewaga Y and Inaba S. 2012. The anamorphic genus Calcarisporiella is a new member of the Mucoromycotina. Mycoscience 53:256-260. https://doi.org/10.1007/S10267-011-0160-1 [ Links ]

Hoog GS. 1974. The genera Blatobotrys, Sporothrix, Calcarisporium and Calcarisporiella Gen. Nov. Studies in Mycology 7:70-73. [ Links ]

Hoyos L, Duque G y Orduz S. 2008. Antagonismo in vitro de Trichoderma spp. sobre aislamientos de Sclerotinia spp., y Rhizoctonia spp. Revista Colombiana de Ciencias Hortícolas 2: 76-86. https://doi.org/10.17584/rcch.2008v2i1.1175 [ Links ]

James T, Marino J, Perfecto I and Vandermeer J. 2016. Identification of putative coffee rust micoparasites via single-molecule DNA sequencing of infected pustules. Applied and Environmental Microbiology 83: 631-639. DOI: 10.1128/AEM.02639-15 [ Links ]

Mahfund MC, Mior AZ, Meon S and Kadir J. 2006. In Vitro and in Vivo Tests for Parasitism of Verticillium psalliotae Treschow on Hemileia vastatrix Berk and Br. Malaysian Journal of Microbiology. 2:46-50. Disponible en línea en: http://www.myjurnal.my/public/article-view.php?id=10291Links ]

Martins JS, Soares CA, Medeiros VH, Santos CV, Pozza EV. 2015. Contribution of host and environmental factors to the Hyperparasitism of coffee rust under field conditions. Australasian Plant Pathology 44:605-610. https://doi.org/10.1007/s13313-015-0375-2 [ Links ]

Moguel P y Toledo V. 1996. El café en México, ecología, cultura y sustentabilidad. Ciencias 43:40-51. [ Links ]

Molina RE, Morales RR, Valenzuela FE, y Vives GI. 2012. Caracterización morfológica de Acremonium sp. asociado a Neonectria fuckeliana en Pinus radiata en Chile. Boletín de Micología 27: 32-38. http://dx.doi.org/10.22370/bolmicol.2012.27.2.883 [ Links ]

OIC. 2015. Informe sobre el brote de la roya del café en Centroamérica y Plan de acción para combatirla. Organización Internacional del Café. Disponible en línea: http://www.ico.orgLinks ]

Rolz AC, De León LR y Paniagua O. 2013. Evidencia de un antagonismo in vitro de especies de Trichoderma contra Hemileia vastatrix (roya del café). Centro de Ingeniería Bioquímica, Instituto de Investigaciones. Universidad del Valle de Guatemala. Revista 25 de la Universidad del Valle de Guatemala. Disponible en línea: http://uvg.edu.gt/publicaciones/revista/volumenes/numero25/8_evidencia%20de%20un.pdfLinks ]

Sandoval S y López OM. 2001. Hiperparasitismo de Trichoderma harzianum, T. viridae y T. pseudokoningii sobre diferentes hongos fitopatógenos. Revista de Fitosanidad, 5:41-44. Instituto de Investigaciones de Sanidad Vegetal. Playa, Ciudad La Habana, Cuba. Disponible en línea: http://www.redalyc.org/articulo.oa?id=209118258010Links ]

Servicio Meteorológico Nacional. (2000). Normales climatológicas. Disponible en línea: http://smn.cna.gob.mxLinks ]

Schieber E. 1972. Economic impact of coffee rust in Latin America. Annual Review of Phytopathology 10:491-510. https://doi.org/10.1146/annurev.py.10.090172.002423 [ Links ]

Shyang CR, Huang CC, Li CJ and Tsay GJ. 2017. Evaluation of characteristics of Simplicillium lanosoniveum on pathogenicity to aphids and in vitro antifungal potency against plant pathogenic fungi. International Journal of Environmental & Agriculture Research 3:55-61. Disponible en línea: http://www.ijoear.com/Paper-January-2017/IJOEAR-JAN-2017-7.pdfLinks ]

Spencer DM and Atkey PT. 1981. Parasitic effects of Verticillium lecanii on two Rust fungi. Transactions of the British Mycological Society. 77:535-542. https://doi.org/10.1016/S0007-1536(81)80101-5 [ Links ]

Zambolim L, Vale FXR, Pereira A e Chaves G. 1997. Café (Coffea arabica L.), Controle de Doenças. In: Vale FXR, Zambolim L. (eds.). Controle de Doenças de Plantas: Grandes Culturas, vol. 1. Suprema Gráfica e Editora, Visconde do Rio Branco, Brasil. 83-40 pp. [ Links ]

Zare R and Gams W. 2001. A revision of Verticillium section Prostata. IV. The genera Lecanicillium and Simplicillium gen. Nov. Nova Hedwigia 73:1-50. DOI: 10.1127/nova.hedwigia/71/2001/1 [ Links ]

Zare R, Gams W and Culham A. 2000. A revision of Verticillium sect. Prostata I. Phylogenetic studies using ITS sequences. Nova Hedwigia 71:465-480. Disponible en línea: https://www.cabdirect.org/cabdirect/abstract/20023073040Links ]

Zare R, Gams W and Culham A. 2000. A revision of Verticillium sect. Prostata I. Phylogenetic studies using ITS sequences. Nova Hedwigia 71:465-480. Disponible en línea: https://www.cabdirect.org/cabdirect/abstract/20023073040Links ]

Received: August 02, 2017; Accepted: December 11, 2017

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