Response of Strawberry Cultivars: Camarosa, Candonga and Ventana to Inoculation with Isolates of Macrophomina phaseolina

1 Response of Strawberry Cultivars: Camarosa, Candonga and Ventana to Inoculation with Isolates of Macrophomina phaseolina M. Avilés, S. Castillo, C. Borrero and M.L. Castillo Departamento de Ciencias Agroforestales, Universidad de Sevilla, Sevilla, Spain T. Zea-Bonilla and R.M. Pérez-Jiménez IFAPA Centro de Churriana, Malaga, Spain Keywords: cultivar susceptibility, Fragaria × ananassa, pathogen virulence Abstract The presence of the soil-borne fungus Macrophomina phaseolina which causes crown and root rot of strawberries has recently been reported in Huelva, Spain. The response toward this pathogen of the cultivars: ‘Camarosa’, ‘Candonga’ and ‘Ventana’ (the most common strawberry cultivars planted in this area) was characterized by artificial inoculation under greenhouse conditions. Six isolates were used in two pathogenicity assays with different inoculation methods: i) by inserting a fungus-colonized toothpick into each crown and ii) by pouring a sclerotia suspension into the pots. The areas under the disease progress curves were used as a disease severity measure for statistical comparisons. For the toothpick inoculation method there were no differences on severity between cultivars or isolates. For the sclerotia suspension inoculation method, results indicate that characterised cultivars manifest different susceptibility to crown and root rot caused by M. phaseolina depending on the isolate. INTRODUCTION Charcoal rot of strawberry (Fragaria × ananassa Duchesne) caused by Macrophomina phaseolina (Tassi) Goidanich has been known for a long time in a variety of production areas in Egypt, France and Illinois (USA) (Maas, 1988). Recently, the disease has been described in Florida (USA) (Mertely et al., 2005), Israel (Zveibil and Freeman, 2005) and in Huelva (Spain) (Avilés et al., 2007). In infected plants, the development of new roots and runners is limited, whilst in advanced states of the disease, the plants wilt, become stunted and die: necrotic roots and crown rot may also be observed. Conducive conditions for the disease are elevated temperatures (optimal growth of the fungus is at 35-39°C) and water-stressed plants. M. phaseolina has a wide host range, amongst these soybean and sunflowers are important hosts. The fungus forms black sclerotia (100-200 μm in diameter) within infected tissues, which permits it to survive on plant debris or in soil under the worst conditions for long periods. Conidia produced into picnidia may also play an important role in the dispersal of the pathogen (Holliday and Punithaligam, 1970). Soilborne diseases are difficult to control. Control of charcoal rot of strawberries has been widely performed in the past by soil fumigation using methyl bromide. Following the phase-out of methyl bromide, new chemicals for soil fumigation to control disease are under evaluation. However, alternative chemicals also have negative effects on human health and imply important alterations to the environment such as soil fertility or water quality. Therefore the use of these chemicals must be, as least, broadly reduced in order to be sustainable. In this context, non-chemical approaches must be considered 2 and developed. The use of strawberry plants resistant or tolerant against this and other soilborne pathogens, as a part of integrated control strategies, is a realistic alternative that must be seriously take into account. Breeding programmes towards resistance against important soilborne disease are in progress in different countries (Faedi et al., 2002). However, to our knowledge, the response of different strawberry germplasm against M. phaseolina has not been studied. This work summarised a study initiated to characterise the response of the most important strawberry cultivars used in Huelva; ‘Camarosa’, ‘Candonga’ and ‘Ventana’ when artificially inoculated with the pathogen. Further considerations in relation to their response should be taken into account for breeding programmes. MATERIALS AND METHODS Isolates. At the end of the 2006 season (May-June) collapsed and dying strawberry plants were sampled in four strawberry fields located in Huelva (Southern Spain). Cut crowns of symptomatic plants revealed dark brown necrotic areas on the margins and along the woody vascular ring. Roots of affected plants were also shown to be necrotic. Six single sclerotium isolates of M. phaseolina developed from surface-disinfested symptomatic tissues and further identified by conventional and molecular methods were selected for inoculations (Table 1). Inoculation methods. Two different inoculation methods were used for characterizations: by inserting a fungus-colonized toothpick into each crown and by pouring a sclerotia suspension into pots (Mertely et al., 2005). For the toothpick-method, M. phaseolina isolates were grown on corn meal agar at 24ºC for 4 days and allowed to colonize sterile wooden toothpicks placed on the medium for an additional 5 days. Prior to use, the toothpicks were sterilized by autoclaving twice in distilled water and a third time in V8 juice. One fungus-colonized toothpick was used per plant. For the sclerotia-method, a sclerotia suspension of each isolate was obtained blending a 7 days-PDA growing colony in 100 ml of sterilized water. 50 ml of suspension, previously adjusted at 1.6 x 103 sclerotia ml-1, was used per plant. Strawberry plants. Strawberry runner plants growing in 1-L pots of coir fibre substrate were used for inoculations. For both methods, six plants of each cultivar were inoculated with each isolate whilst six non-inoculated plants treated similarly were left as controls. After inoculation, plants were incubated in a greenhouse in a randomised complete block design with three replicates of two plants each. For fertilization 3 g/L Osmocote Pro (18+10+11+2MgO+Te, Scotts, Heerlen, The Netherlands) was added per pot. The assay was carried out from July to October 2006. Severity assessment. Disease severity was monitored once per week for two months after inoculation and was scored based on a symptom severity scale where: 0 = asymptomatic plants; 1 = infected plants (leaves chlorotic or wilted); 2 = dead plants. Disease severity was expressed as proportion of the maximum possible disease severity. The area-under-the-disease-progress-curve-standardized (AUDPCs) per plant was calculated by disease severity integrated between symptoms onset and bioassay final time and dividing by the total epidemic time (days). Statistical analysis. Data collected from the trial were analyzed with Statgraphics Plus, Version 6 (SGS, 1999). For each inoculation method, the effect of M. phaseolina isolate and strawberry cultivar on AUDPCs was analyzed with ANOVA. Significant means were compared by Fisher LSD test (P<0.05). 3 RESULTS AND DISCUSSION After 6 days, the toothpick-inoculated plants of different cultivars developed wilting and petiole necrosis in one or more leaves for all isolates. Neither the cultivar nor the isolate showed significant effect on severity. All non-inoculated control plants remained healthy. After 27 days, sclerotia-inoculated plants with all different isolates manifested wilting and petiole necrosis in one or more leaves. Furthermore, after 34 days, when inoculated with the isolate CH 764, ‘Camarosa’, ‘Ventana’ and ‘Candonga’ plants died at the rate of 40%, 100% and 67% respectively. For sclerotia-inoculated plants, isolate and isolate x cultivar interaction showed significant effect on severity (Table 2). For the isolate CH 764 ‘Camarosa’ plants were more tolerant than the other cultivars. Therefore, results indicate that characterised cultivars manifest different susceptibility to crown and root rot caused by M. phaseolina depending on the isolate. Literature cited Avilés, M., Castillo, M.L., Bascón, J., Zea-Bonilla, T., Martín-Sánchez, P.M. and Pérez- Jiménez, R.M. 2007. First report of Macrophomina phaseolina causing crown and root rot of strawberry in Spain. New Disease Report. Vol. 15. Holliday, P. and Punithaligam, E. 1970. CMI Descriptions of Pathogenic Fungi and Bacteria nº 275. Macrophomina phaseolina. CAB International, Wallingford, GB. Faedi, W., Mourgues, F., and Rosati, C. 2002. Strawberry breeding and varieties: situation and perspectives. Acta Hort. 567:51-59. Maas, J. 1998. Compendium of Strawberry Diseases. The American Phytopathological Society, St. Paul, MN, USA. Mertely, J., Seijo, T. and Peres, N. 2005. First report of Macrophomina phaseolina causing a crown rot of strawberry in Florida. Plant Dis. 89:434. Zveibil, A. and Freeman, S. 2005. First report of crown and root rot in strawberry caused by Macrophomina phaseolina in Israel. Plant Dis. 89:1014. Tables Table 1. Characteristics of isolates of Macrophomina phaseolina used in this study. Code Host Cultivar Date of isolation Collection CH 763 Camarosa May 2006 IFAPA (Málaga) CH 764 Camarosa May 2006 IFAPA (Málaga) F-1806 Advanced selection Jun 2006 EUITA (Sevilla) F-91 Camarosa May 2006 EUITA (Sevilla) G-C Coral Jun 2006 EUITA (Sevilla) G-V Advanced selection Jun 2006 EUITA (Sevilla) 4 Table 2. Analyses of variance (ANOVA) of AUDPCsa for plants inoculated by irrigation with a sclerotia-suspension of Macrophomina phaseolina. Source df Mean squareb Isolate 5 1293.27** Cultivar 2 272.673 Block 2 16.99 Isolate x Cultivar 10 160.04* Error 82 71.86 a AUDPCs : area under disease progress curve standardized. Disease severity scale in strawberry plants infested with M. phaseolina: 0 = asymptomatic plants, 1 = symptomatic plants, 2= dead plant. Data for AUDPCs were transformed for analysis with the arcsine √x. b * and ** represent significant effects at the P = 0.05 and P=0.01 level of probability, respectively. Figure ISOLATE CH 763 CH 764 F-1806 F-91 G-C G-V AUDPCs 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 Ventana Candonga Camarosa a a ba a a a a a a a aa a a a a a Figure 1 Cultivar effect for six Macrophomina phaseolina isolates on area under disease progress curve standardized (AUDPCs). Disease severity scale in strawberry plants infested with M. phaseolina (sclerotia-method): 0 = asymptomatic plants, 1 = symptomatic plants, 2= dead plant. Data for AUDPCs were transformed for analysis with the arcsine √x. For each isolate, symbols with the same letter are not significantly different according to LSD test at P<0.05.