Physiological races of Fusarium oxysporum f.sp. melonis in Tunisia
Hela Chikh-Rouhou, Rafika Sta-Baba, Chadha Ayed, Sabeh Belgacem, Naima Boughalleb & Mejda Cherif Phytoparasitica ISSN 0334-2123 Phytoparasitica DOI 10.1007/s12600-013-0321-1
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Author's personal copy Phytoparasitica DOI 10.1007/s12600-013-0321-1
Physiological races of Fusarium oxysporum f.sp. melonis in Tunisia Hela Chikh-Rouhou & Rafika Sta-Baba & Chadha Ayed & Sabeh Belgacem & Naima Boughalleb & Mejda Cherif
Received: 11 March 2013 / Accepted: 23 June 2013 # Springer Science+Business Media Dordrecht 2013
Abstract Fusarium wilt of melon, caused by Fusarium oxysporum f.sp. melonis (Fom), is an important disease; races of the pathogen were identified by inoculating differential standard host cultivars. A total of ten isolates that were obtained from 23 fields located in four different geographical regions were identified as pathogenic. Results indicate that all four known Fom races, namely, 0, 1, 2 and 1.2, were found in north and middle Tunisia. Race 1.2 was the most prevalent. Keywords Fusarium wilt . Melon . Pathogenic isolates
Introduction Fusarium oxysporum f.sp. melonis Snyder & Hans. (Fom) is specific to melon and it is responsible for one of the most important infectious diseases in this fruit species (Suarez-Estrella et al. 2004). Four races of the pathogen (0, 1, 2 and 1.2) have been defined according H. Chikh-Rouhou (*) : N. Boughalleb Institut Supérieur Agronomique de Chott-Mariem, 4042 Sousse, Tunisia e-mail:
[email protected] R. Sta-Baba : S. Belgacem Centre Régional des Recherches en Horticulture et Agriculture Biologique, Chott-Mariem 4042, Tunisia C. Ayed : M. Cherif Institut National Agronomique de Tunis, 1082 Cité Mahrajen, Tunisia
to the host resistance genes overcome by variants of the pathogen (Risser et al. 1976). Race 1.2 is further subdivided into race 1.2 Y, which causes yellowing, and race 1.2 W, which causes wilting without previous yellowing. Race 0 induces disease on melon genotypes that lack Fom resistance genes. Two dominant, independently-inherited resistance genes (Fom-1 and Fom-2) provide resistance to races 0 and 2, and races 0 and 1, respectively (Risser et al. 1976). The presence of both genes confers high resistance to races 0, 1, and 2 (Messiaen et al. 1962). Resistance to race 1.2 is complex and appears to be controlled by multiple recessive genes (Chikh-Rouhou et al. 2007, 2008; Herman & PerlTreves 2007; Perchepied et al. 2005). Partial resistance was found in several Far Eastern lines such as Ogon 9, and was introgressed into the cultivar 'Isabelle' (Perchepied & Pitrat 2004) from which the two doubled-haploid resistant lines Nad-1 and Nad-2 were derived (Ficcadenti et al. 2002) with resistance against Fom race 1.2, confirming its polygenic nature. More recently Chikh-Rouhou et al. (2010, 2011) found resistance in Far Eastern and Iberian lines, and showed that this polygenic resistance is either dominant or recessive and four to eight factors are involved in the resistance to race 1.2 in these lines. The determination of the predominant Fom races in certain geographical melon-growing regions was considered of substantial significance both for combating the disease and for breeding purposes. The aim of this study was to determine, by the use of differential cultivars, the occurrence of Fom races in the main melonproducing areas in Tunisia.
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For inoculation, a root-dip method as described previously (Chikh-Rouhou et al. 2004; Latin & Snell 1986) was used. Following 21 days of growth in pots with steam-sterilized soil–compost substrate, melon seedlings at the first leaf stage were removed and their roots were washed with tap water, and then dipped in conidial suspension (106 conidia ml-1) of the respective Fom isolates for 15 min. Control plants were dipped in sterilized distilled water. Following inoculation, the plants were transplanted to 10-cm-diam pots (one plant per pot) with sterilized soil substrate and kept under greenhouse conditions at 25–28°C. The experimental design was a randomized complete block design; four replications were used per treatment with four plants per replication. Plants were examined every 5 days after inoculation, and the number of yellowed, wilted or dead seedlings was recorded. Symptom severity was assessed on plants on a rating scale from 0 to 4 using the following criteria: (0) no symptoms, (1) beginning of yellowing or wilting on leaves, (2) leaves heavily affected, (3) stem standing and leaves completely wilted, and (4) death of plant. Symptoms were recorded over the 4-week period following the first appearance of symptoms. Plants scored with 0 or 1 were considered resistant, whereas plants scored with 2, 3 and 4 were considered susceptible (Chikh-Rouhou et al. 2007, 2010).
Materials and methods Fungal isolates Diseased melon plants showing symptoms were collected from commercial fields in Tunisia. The pathogen was isolated from diseased plants by placing plant tissues from the lower part of the stem, after surface disinfection with 1% sodium hypochlorite for 2 min, on potato dextrose agar (PDA) media and incubated at 27°C for 3 to 5 days. Then, single spore cultures were derived and maintained on PDA. Fusarium oxysporum was recognized on the basis of morphological characters (Nelson et al. 1983). For inoculum preparation, Fom isolates were grown in potato dextrose broth media in Erlenmeyer flasks for 10 days at 25°C, in an orbital shaker incubator. Spore concentration was determined using a hemocytometer and adjusted to the appropriate concentration of 106 conidia ml-1 by dilution with sterile distilled water. For fungal testers we used the four Fom races, kindly provided by Dr. Gonzaléz-Torres from Spain. Race determination Race determination virulence tests were performed using four differential melon varieties provided by Dr. J. M. Alvarez from Spain. Ten isolates were tested on cv. ‘Charentais T’ (susceptible to all known races), ‘Charentais Fom1’ (resistant to races 0 and 2), ‘Charentais Fom2’ (resistant to races 0 and 1) and ‘Isabelle’ (partially resistant to race 1.2).
Table 1 Race determination of Tunisian isolates of Fusarium oxysporum f.sp. melonis collected from different areas of melon production in Tunisia Isolate
Site of origin
Disease index y (%)
Differential melon cultivars Charentais T
Charentais Fom1
Charentais Fom2
Isabelle
Race
FO1
Bizerte
Sz
R
R
R
0
41ax
FO2
Bizerte
S
S
R
R
1
53b
FO3
Bizerte
S
S
S
R
1.2
75c
FO4
Beja
S
S
S
R
1.2
78c
FO5
Beja
S
R
R
R
0
43a
FO6
Beja
S
R
S
R
2
51b
FO7
Beja
S
S
S
R
1.2
81c
FO8
Kairouan
S
R
R
R
0
40a
FO9
Kairouan
S
R
S
R
2
50b
FO10
Monastir
S
S
S
R
1.2
85c
z
S: susceptible (scored as 2, 3 or 4); R: resistant (scored as 0 or 1) on a scale where 0= plants without symptoms; and 4= dead plant
y
Disease incidence is calculated on the susceptible cultivar ‘Charentais T’
x
Means followed by the same letter do not differ significantly according to the LSD test (P≤0.05)
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Disease incidence (DI) was calculated according to the following equation (Demir et al. 2006): DI(%)=((0*n0)+ (1*n1)+(2*n2)+(3*n3)+(4*n4))*100/(n*4) Where n0 was the number of plants having no symptoms, n1 was the number of plants having disease score 1; n2 was the number of plants having disease score 2; n3 was the number of plants having disease score 3; n4 was the number of dead plants; and n was the total number of plants; 4 was the highest value on the scale. Data analysis Data were subjected to ANOVA analysis. Means were compared by least significant difference (LSD) test at P = 0.05.
Results and discussion The determination of the predominant Fom races in certain geographical melon-growing regions was considered of substantial significance both for combating the disease and for breeding purposes. On the basis of their morphological characters, ten isolates originating from melon plants grown in Tunisia were classified as F. oxysporum. The variable pathogenicity of the examined isolates to differential melon cultivars and their regional origin are presented in Table 1. From the ten isolates used in race determination tests, all the isolates were pathogenic on the cv. ‘Charentais T’, which is susceptible to all known races. One isolate (FO2) was avirulent on cv. ‘CharentaisFom-2’ and was classified as race 1. The isolates FO6 and FO9 were avirulent on cv. ‘CharentaisFom-1’ and were classified as race 2. Four isolates (FO3, FO4, FO7 and FO10) were avirulent on cv. ‘Isabelle’ but virulent on the other differential cultivars and were classified as race 1.2. The remaining three isolates (FO1, FO5 and FO8) were pathogenic only on cv. ‘Charentais T’ and were classified as race 0. Race 1.2 was the most common among the tested isolates. This latter race causes serious damage to melon production worldwide, because it is the most virulent race (Chikh-Rouhou et al. 2010, 2011). The Spanish isolates tested were more virulent than the Tunisian ones, giving a disease index on the susceptible cv. ‘Charentais T’ of 51, 62, 70 and 91% for races 0, 1, 2 and 1.2, respectively. Fusarium wilt disease is best controlled in melons through the use of resistant varieties. However, Fom
race 1.2 is virulent to melon cultivars possessing the specific independent resistance genes Fom-1 and Fom-2 (Chikh-Rouhou et al. 2008; Zink & Thomas 1990). Variation in aggressiveness among the isolates was evident and relevant to the pathogen race (Table 1). This was more pronounced in the case of inoculation with isolates characterized as race 1.2. A tendency for race 1.2 to be the most virulent – with the highest disease incidence and for race 0 to be the least aggressive, was observed. To contribute to the development of efficient measures for the control of Fusarium wilt in the susceptible local cultivars, phenotypic selection procedures and genetic analyses of a considerable number of Fom isolates should be conducted in future studies. Acknowledgments The authors express their sincere thanks to Dr. J. M. Alvarez from CITA (Centro de Investigacion y Tecnologia Agroalimentaria), Spain, for providing seeds from the differential cultivars used in this study. We are grateful also to Dr. R. González-Torres, for providing fungal testers.
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