Plant Soil (2012) 358:75–89 DOI 10.1007/s11104-012-1205-8
REGULAR ARTICLE
Comparative root colonisation of strawberry cultivars Camarosa and Festival by Fusarium oxysporum f. sp. fragariae Xiangling Fang & John Kuo & Ming Pei You & Patrick M. Finnegan & Martin John Barbetti
Received: 20 November 2011 / Accepted: 2 March 2012 / Published online: 28 March 2012 # Springer Science+Business Media B.V. 2012
Abstract Background and aims Strawberry (Fragaria x ananassa) is a high-value crop worldwide. Fusarium oxysporum f. sp. fragariae causes rapid wilting and death of strawberry plants and severe economic losses worldwide. To date, no studies have been conducted to determine colonisation of either susceptible or resistant strawberry plants by F. oxysporum f. sp. fragariae, or whether plant colonisation by F. oxysporum f. sp. fragariae differs between susceptible and resistant cultivars. Methods Colonisation of strawberry plants by a pathogenic isolate of F. oxysporum f. sp. fragariae was
Responsible Editor: Jesus Mercado-Blanco. X. Fang : M. P. You : P. M. Finnegan : M. J. Barbetti School of Plant Biology, Faculty of Natural Agricultural Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia J. Kuo The Centre for Microscopy, Characterisation and Analysis, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia M. J. Barbetti (*) The UWA Institute of Agriculture, Faculty of Natural Agricultural Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia e-mail:
[email protected]
examined both on the root surface and within root tissue of one resistant cv. Festival and one susceptible cv. Camarosa using light and scanning electron microscopy from 4 h to 7 d post inoculation (pi). Results Resistant cv. Festival significantly impeded the spore germination and penetration from 4 to 12 hpi and subsequent growth and colonisation by this pathogen until 7 dpi compared with susceptible cv. Camarosa. At 7 dpi, fungal colonisation in resistant cv. Festival remained mainly confined to the epidermal layer of the root, while in susceptible cv. Camarosa, hyphae not only had heavily colonised the cortical tissue throughout but had also colonised vascular tissues. Conclusions This study demonstrates for the first time that resistance of a strawberry cultivar to F. oxysporum f. sp. fragariae is a result of impedance of pathogen growth and colonisation both on the plant surface and within host tissues. Resistance mechanisms identified in this study will be of high value for breeding programmes in developing new disease-resistant cultivars to manage this serious strawberry disorder. Keywords Fragaria x ananassa . Fusarium wilt . Hyphal penetration . Vascular tissue
Introduction Fusarium oxysporum is well represented among communities of soil-borne fungi, in all soil types across the
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world (Burgess 1981; Fravel et al. 2003). It is considered to be a normal constituent of the fungal rhizosphere community of plants (Fravel et al. 2003; Gordon and Martyn 1997). F. oxysporum is saprophytic/necrotrophic and grows and survives for long periods on organic matter in soil and in the rhizosphere of many plant species (Fravel et al. 2003; Garrett 1970). Wilt-inducing isolates of F. oxysporum are host-specific fungal pathogens with worldwide distribution, and have been divided into more than 120 different formae speciales (f. spp.) according to their host range across a wide range of plant families (Fravel et al. 2003; Michielse and Rep 2009). F. oxysporum infects host plants by penetrating plants through roots and it is responsible for severe damage and yield losses on many economically important plant species (Fravel et al. 2003; Michielse and Rep 2009). F. oxysporum f. sp. fragariae, the causal agent of Fusarium wilt on strawberry, is a serious threat to commercial strawberry production worldwide. Fusarium wilt on strawberry was first reported in Australia in 1965 (Winks and Williams 1965) and subsequently reported in Korea (Kim et al. 1982; Nagarajan et al. 2006), China (Zhao et al. 2009), Spain (Arroyo et al. 2009) and the USA (Koike et al. 2009). F. oxysporum f. sp. fragariae penetrates strawberry plants through roots, severely affecting roots and crowns, and resulting in rapid wilting and eventually death of strawberry plants (Fang et al. 2011a, b; Koike et al. 2009). Management of Fusarium wilt on strawberry plants is mainly through chemical soil fumigation and/or resistant cultivars worldwide. However, broad-spectrum pre-planting fumigants, such as methyl bromide, can be environmentally damaging (Fravel et al. 2003), and the phase-out of methyl bromide has not only forced development of alternative fumigants, but has fostered keen interest in alternative means for management of Fusarium wilt (Easterbrook et al. 1997; Fang et al 2012). Identifying and deploying resistant cultivars is considered to be the most cost effective and environmentally sustainable strategy for control of Fusarium wilt (Fravel et al. 2003; MacKenzie et al. 2006; Particka and Hancock 2005). Studies on plant colonisation by pathogens on plant cultivars with different levels of susceptibility/resistance have contributed towards development of new diseaseresistant cultivars and more effective disease-control strategies. This is especially so for pathogens that are difficult to control, such as Sclerotinia on Brassicas (Garg et al. 2010) and Phytophthora on subterranean clover (Ma et al. 2010), which suggests similar potential
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for control of Fusarium wilt on strawberry. Susceptibility of plants to a pathogen is defined as a compatible interaction, while resistance of plants to a pathogen is defined as an incompatible interaction. Colonisation of plants by different formae speciales of F. oxysporum other than F. oxysporum f. sp. fragariae has been studied in terms of susceptible plants on several different host species, including tomato plants by F. oxysporum f. sp. lycopersici (e.g., Jonkers et al. 2009; Lagopodi et al. 2002), and cotton plants by Fusarium oxysporum f. sp. vasinfectum (e.g., Dowd et al. 2004; Rodríguez-Gfilvez and Mendgen 1995). In contrast, studies of plant colonisation by different formae speciales of F. oxysporum in terms of resistant plants have been hampered due to the lack of plant material with resistance against some Fusarium wilt disorders (e.g., on cotton; Dowd et al. 2004). To date, colonisation of strawberry plants by F. oxysporum f. sp. fragariae has not been previously studied in relation to either susceptible or resistant strawberry plants, and whether plant colonisation by F. oxysporum f. sp. fragariae differs between susceptible and resistant strawberry cultivars remains unknown. A previous study by Fang et al. (2012) found that strawberry cv. Camarosa was the most susceptible cultivar to F. oxysporum f. sp. fragariae, while strawberry cv. Festival was the most resistant cultivar. Therefore, this paper reports studies conducted to determine, for the first time, the colonisation of both the root surface and within root tissues of susceptible and resistant strawberry plants by F. oxysporum f. sp. fragariae.
Materials and methods Fungal isolate A single-spore isolate of F. oxysporum f. sp. fragariae WUF-ST-FO51, obtained from severely affected strawberry plants collected in 2008 from a major commercial strawberry field (Wanneroo, Western Australia, Australia; latitude/longitude: −31.8/115.8), was used throughout this study. This isolate has been deposited into the Western Australian Culture Collection Herbarium maintained at the Department of Agriculture and Food Western Australia (Accession No. WAC13497). This isolate was previously reported to be highly pathogenic to strawberry plants (Fang et al. 2011b). The isolate was stored as colonised filter paper pieces that were dried at room temperature (approximately 20–22 °C).
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Strawberry cultivars and tissue culture Strawberry cultivars Camarosa and Festival were purchased as certified commercial runners (Toolangi Certified Strawberry Runner Grower’s Co-Op Ltd, Victoria, Australia), and were maintained in a glasshouse growing in pots with potting mix at the University of Western Australia, Australia. In earlier studies, cv. Camarosa was found to be susceptible to Fusarium oxysporum f. sp. fragariae, and cv. Festival was found to be resistant (Fang et al. 2012). Responses of these two strawberry cultivars to F. oxysporum f. sp. fragariae were reconfirmed by the highly pathogenic isolate WUF-ST-FO51 using the same inoculation method and growth conditions as described previously (Fang et al. 2012), consistently showing cv. Camarosa as highly susceptible and cv. Festival as highly resistant (Fig. 1). A tissue culture system was developed to aseptically produce seedlings from resistant and susceptible strawberry cultivars for this study based on methods described by Bhatt and Dhar (2000) and Sakila et al. (2007). Seedlings of the two strawberry cultivars were removed from tissue culture tubes, and washed thoroughly with sterilised DI water before use. Inoculum preparation Pieces of filter paper colonised by F. oxysporum f. sp. fragariae WUF-ST-FO51 were cultured on to potato dextrose agar (PDA) plates and maintained at 22 °C
Fig. 1 Two strawberry cultivars, susceptible cv. Camarosa (a) and resistant cv. Festival (b), used in this study. FoF, strawberry plants inoculated by F. oxysporum f. sp. fragariae; CK, control plants which were inoculated by DI water. Photos were taken at 4 weeks after inoculation
under continuous fluorescent light. After 2 weeks, the fungal culture on each plate was flooded with 10 mL of sterilised DI water and the agar surface was gently rubbed with a bent glass rod. Spore suspension was then filtered through four layers of Miracloth™ (Calbiochem, Merck Pty Ltd, Victoria, Australia) to remove any agar or mycelia segments. The Fusarium spores in the filtrate were washed three times by centrifugation (11000×g, 20 min) and re-suspended in sterilised DI water. Concentration of the spore suspension was adjusted to 1×106 spore mL−1 by diluting with sterilised DI water using a haemocytometer (SUPERIOR®, Berlin, Germany). Plant inoculation and growth conditions Seedlings of both strawberry cultivars were inoculated by dipping roots in the spore suspension of F. oxysporum f. sp. fragariae for 10 min. Controls were inoculated by dipping roots in DI water for 10 min. At the same time, drops (20 μL) of spore suspension were deposited onto glass slides to compare the spore germination in DI water with what occurred on roots. After inoculation, both seedlings and glass slides were placed in Petri dishes containing moistened filter paper and sealed with Parafilm® to study the early stages of spore germination on roots and in DI water, and penetration of germinated spores on roots. In order to protect roots from light, the lower halves of Petri dishes with seedlings were wrapped into aluminum foil. Petri dishes were placed in an upright position (but at an angle of 60°) in a
A
B
CK
FoF
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growth cabinet (22 °C, 16 h photoperiod, 60 % relative humidity). Inoculated seedlings were also transplanted into pots with sterilised sand and moistened with sterilised DI water for further study of colonisation of roots by F. oxysporum f. sp. fragariae.
sputter coated with gold particles and examined and photographed (Zeiss 1555 VP-FESEM, Carl Zeiss, NSW, Australia). This experiment was repeated twice more.
Sample preparation for light microscopy study
Roots of inoculated and control seedlings of the two strawberry cultivars were collected at 3, 5, and 7 dpi. A total of 6 roots (one from each replicate plant from each treatment) were sampled at each time point and prepared for glycol methacrylate (GMA) biological sampling. Root segments were fixed in 2.5 % (v/v) glutaraldehyde in 0.05 M phosphate buffer (pH 7.0) for 24 h at 4 °C. Fixed samples were kept under vacuum to remove air bubbles inside tissues and then a fresh fix solution applied to samples. Samples were vacuum infiltrated for 6 min (2 min on / 2 min off / 2 min on) at 80 W in a microwave (PELCO BioWave® Microwave Processor, Ted Pella Inc.), and then washed with 0.05 M phosphate buffer for 40 s at 80 W. Samples were then dehydrated in a series of acetone solutions ranging from 30 % to 100 % (dry) acetone for 4 min (2 min on / 1 min off / 1 min on) at 250 W for three times at each concentration. After dehydration, samples were vacuum infiltrated in 1:3, 1:2, 1:1, 2:1 and 3:1 (vol/vol) GAM/acetone and 100 % GMA for 3 min each at 250 W for three times. Samples in 100 % GMA were left on a rotator at 40 rpm for at least 16 h before replacing with the fresh GMA solution and rotating at 40 rpm for another 48 h and embedding in GMA in plastic moulds under vacuum at 65 °C for 24 h. Embedded tissues were sectioned into transversal semithin sections (2 μm) using glass knives on a Sorvall® microtome and sections were mounted on microscope glass slides. The same staining procedures were applied to sections from each sample/treatment. Staining with 0.5 % Toluidine Blue O in benzoate buffer (pH 4.4) enabled excellent differentiation between plant cells and fungal hyphae and this was used to study colonisation processes of fungal pathogen in plant tissues and for the detection of phenolic substances (Pannecoucque and Höfte 2009; Ruzin 1999). Sections were also stained for the detection of other substances such as starch (periodic acid/Schiff’s reagent) (Garg et al. 2010), and callose (0.05 % aniline blue in 0.067 M K2HPO4 buffer, pH 9.0) (Pannecoucque and Höfte 2009). Sections of each sample were then examined and photographed (Axioplan 2 microscope, AxioCam Digital photograph system, Carl Zeiss, NSW, Australia).
Roots of inoculated and control seedlings of the two strawberry cultivars were collected at 4, 8 and 12 hpi, 1, 3, 5 and 7 dpi. A total of six roots (one from each replicate plant from each treatment) were sampled at each time point. Roots were stained with 0.05 % (w/v) Trypan Blue in lactoglycerol solution, and whole, wet mounts were examined and photographed (Axioplan 2 microscope, AxioCam Digital photograph system, Carl Zeiss, NSW, Australia). Spore suspensions deposited on glass slides were used as a comparison. One hundred spores were counted in six random fields of views per root for 6 roots (one from each replicate plant from each treatment; i.e. 600 spores per treatment) and the same procedure was repeated for spore suspensions deposited on glass slides. Spores were considered to have germinated if the length of the germ tube was at least half the length of the spore. Percentage of germinated spores and percentage of germinated spores that had penetrated the root were determined. This experiment was repeated twice more. Sample preparation for scanning electron microscopy study Roots of inoculated and control seedlings of the two strawberry cultivars were collected at 4, 8 and 12 hpi. A total of six roots (one from each replicate plant from each treatment) were sampled at each time point. Root segments were fixed in 2.5 % glutaraldehyde (v/v) in 0.05 M phosphate buffer pH 7.0 for 24 h at 4 °C. Samples were vacuum infiltrated for 6 min (2 min on / 2 min off / 2 min on) at 80 W in a microwave (PELCO BioWave® Microwave Processor, Ted Pella Inc.), and then washed with 0.05 M phosphate buffer for 40 s at 80 W. Samples were then dehydrated through a series of ethanol solutions increasing sequentially from 50 % to 100 % (dry) ethanol and subsequently twice with 100 % (dry) ethanol again for 40 s each at 250 W, before being critically point dried (PELCO Critical Point Dryer, Ted Pella Inc.) using liquid carbon dioxide. Samples were
Sample preparation for internal anatomical study
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Proportion of roots colonised by F. oxysporum f. sp. fragariae Roots of inoculated and control seedlings of the two strawberry cultivars were collected at 1, 3, 5 and 7 dpi. A total of 6 roots (one from each replicate plant from each treatment) were sampled at each time point for each treatment. 20–30 hand sections (~1 mm) were cut from each surface sterilised root at each time point for each treatment. Sections of each root were transferred to one PDA plate, and there were 6 plates for the 6 roots at each time point for each treatment. Plates were incubated at 22 °C for 2 days in darkness. Tissue sections on each PDA plate were then examined for the presence of typical mycelial growth and spores of F. oxysporum f. sp. fragariae. Sections of each root on each PDA plate showing typical F. oxysporum f. sp. fragariae mycelial growth and spores were counted, and percentage root colonisation calculated as ½ðtotal sections colonised=total sectionsÞ 100. This experiment was repeated twice more. Statistical analyses Data analyses were conducted using GenStat (11th edition, Lawes Agricultural Trust, Rothamsted Experimental Station, UK, 2011). For all analyses, data from all three repeated experiments (i.e., original and the two repeat experiments) were combined for subsequent calculation of means (n018) and analysed. Analyses of variance were conducted to determine the effects of cultivar and time treatments on spore germination, penetration of germ tube and root colonisation by F. oxysporum f. sp. fragariae. Subsequently, multiple comparisons were made among mean values based on Fisher’s least significant differences (LSD) at P
