Fungal diseases of strawberry and their diagnosis.

C. Garrido; V. E. González-Rodríguez; M. Carbú; A. M. Husaini; J. M. Cantoral

166 10.3. Diagnosis Methods and Field Monitoring of Strawberry Diseases 167 10.3.1. Molecular methods applied to phytopathogenic fungi 167 10.3.2. Past and present diagnosis methods 168 10.3.3. PCR alternatives applied to fungal diagnosis 169 10.3.4. Other molecular techniques used to study fungal pathogens 172 10.3.5. Web-based decision support systems 174 10.3.6. Proteomics advances in strawberry fungal pathogens 181 10.4. Conclusions 184

©CAB International 2016 – for Amjad M.Husaini 10 Fungal Diseases of Strawberry and their Diagnosis Carlos Garrido1, Victoria E. González-Rodríguez1, María Carbú1, Amjad M. Husaini2 and Jesús M. Cantoral1* 1 Departamento de Biomedicina, Biotecnología y Salud Pública, Facultad de Ciencias del Mar y Ambientales, Instituto Universitario de Investigación Vitivinícola y Agroalimentaria (IVAGRO), Universidad de Cádiz, Puerto Real, Spain; 2Centre for Plant Biotechnology, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shalimar, Jammu and Kashmir, India 10.1. Introduction 10.1.1. Modern strawberry, a domesticated species for production 10.1.2. Economic importance of world strawberry production 10.1.3. Pathogen incidence in strawberry production 10.2. The Main Fungal Pathogens of Strawberry 10.2.1. Botrytis cinerea 10.2.2. Colletotrichum spp. 10.2.3. Fusarium oxysporium 10.2.4. Phytophthora spp. 10.2.5. Verticillium spp. 10.2.6. Other genera 10.3. Diagnosis Methods and Field Monitoring of Strawberry Diseases 10.3.1. Molecular methods applied to phytopathogenic fungi 10.3.2. Past and present diagnosis methods 10.3.3. PCR alternatives applied to fungal diagnosis 10.3.4. Other molecular techniques used to study fungal pathogens 10.3.5. Web-based decision support systems 10.3.6. Proteomics advances in strawberry fungal pathogens 10.4. Conclusions 10.1. Introduction 10.1.1. Modern strawberry, a domesticated species for production Over the centuries, men have cultivated different species of the genus Fragaria for 157 157 158 158 159 162 162 163 164 165 166 167 167 168 169 172 174 181 184 strawberry production. The genus Fragaria belongs to the family Rosaceae and consists of more than 28 species, including several subspecies (Garrido et al., 2011). More than 300 years ago, species such as Fragaria chiloensis, Fragaria virginiana, Fragaria vesca and Fragaria moschata were * jesusmanuel.cantoral@uca.es © CAB International 2016. Strawberry: Growth, Development and Diseases (A.M. Husaini and D. Neri) 157 ©CAB International 2016 – for Amjad M.Husaini 158 C. Garrido et al. cultivated across the world for strawberry production. Progressively, these species were subjected to a ‘domestication’ process and became adapted to different climatic regions, developing resistance to regional pests and leading to special fruit characteristics, resulting in the development of new species and subspecies (http:// www.juntadeandalucia.es/organismos/ agriculturapescaydesarrollorural.html). The modern strawberry species Fragaria × ananassa is the most evident result of these processes. This species was developed in France around the middle of the 18th century, by crossing of Fragaria virginiana Duchesne (from North America) and Fragaria chiloensis Duchesne (from South America). After several rounds of crossing between hybrids, a plant similar to the modern Fragaria × ananassa was obtained. This new species, a hybrid, developed larger, more fragrant and tastier red berries, and showed a great capacity for regional adaptation (Maas, 1998). Due to these characteristics, this new species replaced the old ones, especially after it was introduced to America and the rest of Europe. Over several decades, F. × ananassa became the most important species commercially and is now predominantly cultivated for strawberry production worldwide (Garrido et al., 2011). Fragaria × ananassa shows a high level of regional adaptation due to the phenotypic plasticity of its plants. As the original hybrid plants were widely cultivated in different continents, and in more than 50 countries, the regional environmental pressures led to the development of new cultivars. Photoperiod, temperature, humidity, composition of the substratum, ease of obtaining nutrients and the presence of pathogens were some of the different environmental characteristics that contributed to the appearance of different cultivars. Although it is difficult to know the exact number of cultivars present in the world, only a dozen of them constitute the most common varieties used for world strawberry production (Garrido et al., 2009a). 10.1.2. Economic importance of world strawberry production The Statistical Office of The Food and Agriculture Organization (FAO) has released estimates of crop production in the world, classified by commodities × country or by countries × commodity (http://faostat.fao. org). The FAO estimated that the annual world production of strawberry exceeded 7,700,000 t in 2014. This data represented an increase of almost 1,200,000 t in comparison with 2010 (FAO, 2010–2014). Fragaria × ananassa is cultivated in more than 70 countries, with Asia and the Americas being the major producers of strawberry, accounting for 49.7 and 25.2%, respectively (FAO, 2010–2014). The European Union (EU) is third, with 19.2% of world strawberry production. Between 2010 and 2014, China increased its strawberry production, and is currently the country with the highest level of production, with an average of 2,600,000 t per year, relegating the USA, traditionally the most important producer, to second place with only half the production level of China at 1,300,000 t. In the USA, production is concentrated mainly in three states, California, Florida and Oregon, in decreasing order of importance. In the EU, Spain is the most important country, with an average production between 2010 and 2014 of 285,000 t. Spanish production is concentrated in the provinces of Huelva and Cádiz in the south-west of Spain, with a total cultivated area of approximately 7000 ha (FAO, 2010–2014). 10.1.3. Pathogen incidence in strawberry production Strawberry plants, like other commercial crops, can be damaged by environmental, genetic and biological factors, either directly or by interactions between these factors (Garrido et al., 2011). These strawberry health problems lead to significant economic losses to the growers and producer countries. Environmental factors causing ©CAB International 2016 – for Amjad M.Husaini Fungal Diseases of Strawberry damage to the cultivars can be minimized by creating good agricultural practice facilities. Strawberry plants are affected by arthropods, nematodes, fungi, bacteria and viruses. These cause considerable financial losses, running into hundreds of millions of dollars/euros per year (FAO, 2008–2013) (http://www.juntadeandalucia.es/organismos/ agriculturaypesca.html). There are several governmental agencies dedicated to preventing the spread of pathogens during international trade. The two major global agencies are the FAO and the European and Mediterranean Plant Protection Organization (EPPO). The EPPO is an intergovernmental organization, with 50 member countries, responsible for cooperation in plant protection in Europe and the Mediterranean region (http://www. eppo.org). This organization has the aim of helping its member countries prevent the entry or spread of dangerous pests. For this purpose, EPPO identifies pests, makes proposals on the phytosanitary measures that could be taken, and makes available several standards on Pests Risk Analysis databases. Since the 1970s, this organization has periodically published several pest lists that include: ‘List of pests recommended for regulation as quarantine pests’ (A1 and A2 list), ‘Action List’, ‘Alert List’ and ‘List of invasive alien plants’ (Capote et al., 2012). In response to the lists and reports published by this organization, the member countries adopt various different phytosanitary measures and plant protection programmes. We recommend consulting the relevant one at the EPPO website (http://www.eppo.int/ QUARANTINE/quarantine.htm). More than 50 different species of phytopathogenic fungi can infect F. × ananassa cultivars, but not all of them have the same commercial significance (Table 10.1). In relation to quarantine pest lists, in recent years several genera of strawberry fungal pathogens, such as Colletotrichum acutatum, Botrytis cinerea and Phytophthora spp. have been included in the EPPO A2 list (the A2 list includes pests that are locally present in the EPPO region but not widely distributed), but in the last public A2 list from 159 September 2015, only Phytophthora fragariae (specific for strawberry), Verticillium dahliae, Verticillium albo-atrum and Fusarium oxysporum were included (EPPO, 2015). This is not good news, as it implies that Colletotrichum acutatum and B. cinerea are currently widespread throughout Europe. It has therefore become more important to conduct studies on these fungi, as well as making an effort to control the spread of Phytophthora spp. and Verticillium spp. in the EPPO region. 10.2. The Main Fungal Pathogens of Strawberry Strawberry plants are affected by a large number of diseases caused by fungi, bacteria, viruses, nematodes and arthropods. These pathogens cause damage on the leaves, roots, crowns and fruits. Susceptibility of the host plant, the particular pathogen and favourable environmental conditions (temperature, moisture) are the three main factors necessary for the initiation and development of plant disease. In the cultivated strawberry (F. × ananassa), each cultivar differs in its susceptibility to different pathogens and to their races or pathotypes. These races are not evenly distributed around the world, and are often present only in specific strawberry crop regions. Therefore, the damage caused in strawberry crops and the economic losses generated are of different scales for each region (Maas, 2004). The American Phytopathological Society (APS) is the premier society dedicated to the study and control of plant diseases. The APS has published significant breakthroughs in plant pathology, mycology, virology, bacteriology, nematology and related disciplines for more than 100 years. APS members have been responsible for publishing a list of the most common types of damage affecting more than 100 crops, including strawberry cultivation (http://www.apsnet.org/publications/commonnames/Pages/Strawberry. aspx). This list includes more than 67 species of fungal pathogens (Table 10.1), of which 47 species belong to the phylum ©CAB International 2016 – for Amjad M.Husaini 160 C. Garrido et al. Table 10.1. Strawberry fungal pathogens. Genus Species Strawberry disease Referencea Alternaria alternata tenuissima mellea niger cinerea spp. fragariae vexans acutatum Miyamoto et al. (2009) Shafique et al. (2009) Prodorutti et al. (2009) Chiotta et al. (2009) Vallejo et al. (2002) Ruiz-Moyano et al. (2009) Maas et al. (1998) Maas et al. (1998) Garrido et al. (2009a) fragariae Black leaf spot Alternaria fruit rot Armillaria crown rot and root rot Aspergillus fruit rot Botrytis rot fruit; grey mould Cladosporium fruit rot Cercospora leaf spot Cercospora leaf spot Anthracnose on leaves and fruit, crown rot and black spot Anthracnose on leaves and fruit, crown rot and black leaf spot Anthracnose on leaves and fruit; crown rot and black spot Black root rot Gnomonia Hainesia fuckelli destructans earlianum oxysporum sambucinum comari lythri Black root rot Root rot Leaf scorch Fusarium wilt Fruit blotch Leaf blotch; stem end rot Black root rot; Hainesia leaf spot Idriella Macrophomina lunata phaseolina Mucor Mycosphaerella hiemalis mucedo piriformis fragariae Olpidium louisianae brassicae Idriella root rot Macrophomina leaf blight Macrophomina root rot Mucor fruit rot Mucor fruit rot Mucor fruit rot Purple leaf spot; black seed disease Purple leaf spot Olpidium root infection Armillaria Aspergillus Botrytis Cladosporium Cercospora Colletotrichum gloeosporioides fragariae Coniothyrium Cylindrocarpon Diplocarpon Fusarium Pestalotia Penicillium Peronospora Phytophthora longisetula cyclopium expansum frequentans purpurogenum potentillae bisheria cactorum citricola citrophthora cyptogera fragariae megasperma nicotianae Pestalotia fruit rot Penicillium fruit rot Penicillium fruit rot Penicillium fruit rot Fruit rot; fruit blotch Downy mildew; fruit blotch Root rot Leather rot of fruit; Phytophthora crown and root rot Phytophthora crown and root rot Leather rot; Phytophthora crown and root rot Root rot Red stele, red core and root rot Crown rot Leather rot; Phytophthora crown and root rot Chung et al. (2010) Ortega-Morales et al. (2009) Douglas Gubler and Converse (1993) Pertot et al. (2012) Martin et al. (2002) Whitaker et al. (2009) Avis et al. (2009) Hunter et al. (1974) Morocko et al. (2007) Douglas Gubler and Converse (1993) Maas et al. (1998) Maas et al. (1998) Javaid et al. (2009) Hauke et al. (2004) Hauke et al. (2004) Hauke et al. (2004) Ehsani-Moghaddam et al. (2006) Maas et al. (1998) Douglas Gubler and Converse (1993) Maas et al. (1998) Gutierrez et al. (2009) Liu et al. (2007) Redondo et al. (2009) Redondo et al. (2009) Choi et al. (2009) Abad et al. (2008) Nicastro et al. (2009) Haesler et al. (2008) Kong et al. (2009) Abad et al. (2008) Nicastro et al. (2009) Abad et al. (2008) Böszörményi et al. (2009) Continued ©CAB International 2016 – for Amjad M.Husaini Fungal Diseases of Strawberry 161 Table 10.1. Continued. Genus Species Strawberry disease Referencea Phoma lycopersici terrestris Leaf stalk rot Grey sterile fungus root rot Phomopsis Pichia obscurans membranefaciens Phomopsis leaf blight Postharvest rot subpelliculosa Postharvest rot ultimum fragariae Maas et al. (1998) Douglas Gubler and Converse (1993) Nita et al. (2003) Douglas Gubler and Converse (1993) Douglas Gubler and Converse (1993) Triky-Dotan et al. (2009) Lamondia et al. (2005) Rosellinia stolonifer sexualis necatrix Saccharomyces cerevisiae Black root rot Black root rot; anther and pistil blight; Hard brown rot; Rhizoctonia leaf blight Rhizopus rot Rhizopus rot Dematophora crown and root rot (white root rot) Postharvest rot kluyveri Postharvest rot Schizoparme straminea Fruit blotch Sclerotinia Sclerotium sclerotiorum rolfsii fragariae aciculosa fragariaecola malorum Sclerotinia crown and fruit rot Sclerotium rot; southern blight; fruit blotch Septoria hard rot and leaf spot Septoria leaf spot Septoria leaf spot Fruit blotch macularis fragariae albo-atrum dahliae bailli Powdery mildew Stagonospora hard rot Verticillium wilt Verticillium wilt Postharvest rot florentinus Postharvest rot Pythium Rhizoctonia solani Rhizopus Septoria Sphaeropsis Sphaerotheca Stagonospora Verticillium Zygosaccharomyces a Chiba et al. (2009); Liu et al. (2009) Maas et al. (1998) Maas et al. (1998) Pliego et al. (2012) Douglas Gubler and Converse (1993) Douglas Gubler and Converse (1993) Douglas Gubler and Converse (1993) Ren et al. (2010) Errakhi et al. (2009) Maas et al. (1998) Maas et al. (1998) Maas et al. (1998) Douglas Gubler and Converse (1993) Davik et al. (2005) Maas et al. (1998) Larsen et al. (2007) Costa et al. (2007) Douglas Gubler and Converse (1993) Douglas Gubler and Converse (1993) Relevant publications from authors working with the pathogen in the plant pathology field. Ascomycota, ten to the Oomycota, four to the Basidiomycota, one to the Chytridiomycota and five to the Zygomycota. Several species of fungi are capable of causing damage to more than one part of the strawberry plant (leaf, root, crown and/or fruit), whereas other species affect only a specific part of the plant; for example, Colletotrichum acutatum causes anthracnose on leaves and fruits, crown rot and black spot, while B. cinerea causes damage to the fruit only (Table 10.1). Various fungi invade the primary roots and attack/destroy secondary roots, limiting the plant’s ability to take up water and nutrients. Leaf pathogens cause various injuries or overwinter in dead leaves and petioles, and form spores. These spores are disseminated by wind and/or rain and irrigation water, and initiate new infections. Fruit can be affected ©CAB International 2016 – for Amjad M.Husaini 162 C. Garrido et al. by a large number of genera of fungi, with B. cinerea, Phytophthora cactorum and Colletotrichum spp. being the major strawberry fruit disease problems worldwide, resulting in the greatest fruit losses. These also account for the highest amounts of fungicide use for strawberry fruit protection. 10.2.1. Botrytis cinerea B. cinerea Pers.: Fr. is a phytopathogenic ascomycete that causes grey mould in more than 200 crop species worldwide, without any apparent host specificity. In strawberry, this pathogen is the cause of the disease known as ‘Botrytis rot fruit’ (Table 10.1), which causes tremendous losses in the field (expected 80–90% loss of both flowers and strawberries) during rainy and cloudy periods, just before or during harvest and storage. This necrotrophic fungus attacks different organs, such as shoots, leaves, flowers and fruit, and is more destructive on mature or senescent tissue, but usually gains entry to such tissues at the flowering stage. Flowers are usually infected during blossoming, and the pathogen then enters the young fruits at a very early stage of their development. It remains latent for a considerable period before rapidly decomposing the tissues when environmental factors, such as relative humidity, and fruit physiology are optimal. The fungus infects the fruit, causing it to become deformed, dried out, dark and rapidly covered with a powdery layer of spores, which gives a grey appearance. In general, strawberries that are in contact with the ground or with another rotten strawberry or dead leaves in dense foliage are the ones that are commonly affected by this phytopathogenic fungus. The long latency period of the pathogen from early infection until symptoms appear makes grey mould control very difficult. Therefore, the most common method used to control the infection and its spread has been the regular application of fungicides throughout flowering. Another alternative is the use of cultivars resistant to grey mould. The genetic resistance in strawberry to Botrytis infection appears to be multigenic and has a very low general combining ability. There has been little success in breeding and selecting cultivars resistant to this disease (Maas, 2004). A recent study determined the resistance of new cultivars, currently grown in Florida, and advanced selections from the University of Florida’s breeding programme to Botrytis fruit rot (Seijo et al., 2008). The results demonstrated that the cultivars ‘Camarosa’, ‘Florida Radiance’, ‘Florida Elyana’ and advanced selections 99-117 and 99-164 showed good levels of resistance, whereas ‘Camino Real’, ‘Ventana’, ‘Treasure’, ‘Candonga’, ‘Strawberry Festival’ and ‘Sweet Charlie’ were more susceptible. The major difficulty encountered in the fight against this disease is the lack of natural genetic resistance to grey mould in strawberry germplasm, making unsuccessful all attempts to incorporate tolerance to this disease into strawberry lines. The development of biotechnology has provided new opportunities to enhance disease resistance for strawberry breeding. Vellicce et al. (2006) obtained transformed strawberry plants (cultivar ‘Pájaro’) using three defence-related genes: ch5B, encoding a chitinase from Phaseolus vulgaris; and gln2 and ap24, encoding a glucanase and a thaumatin-like protein, respectively, both from Nicotiana tabacum. The results showed that constitutive expression of the bean ch5B gene in this strawberry cultivar was an effective strategy to provide protection against B. cinerea. The authors concluded that the same methodology could be used to introduce resistance into F. × ananassa germplasm. Other studies on transformation of strawberry plants using Agrobacterium-mediated transformation have resulted in the development of transgenic lines expressing glucose oxidase (Jin et al., 2005) and thaumatin-like proteins (Schestibratov and Dolgov, 2005). Both of these transgenic strawberry lines showed a significantly higher level of resistance to grey mould. 10.2.2. Colletotrichum spp. Colletotrichum spp. comprises a diverse range of important phytopathogenic fungi that cause pre- and postharvest crop losses worldwide. Three species have been reported ©CAB International 2016 – for Amjad M.Husaini Fungal Diseases of Strawberry as causal agents of strawberry (F. × ananassa) anthracnose: Colletotrichum acutatum, C. fragariae and C. gloeosporioides (telemorph, Glomerella cingulata) (Denoyes-Rothan et al., 2003) (Table 10.1), which can infect strawberry plants in nurseries, field production, or during harvest and storage of fruits. All three species can be found on all parts of the plant (Denoyes-Rothan et al., 2003), although C. fragariae and C. gloeosporioides are the principal causal agents of anthracnose crown rot, whereas Colletotrichum acutatum has been well documented as the principal causal agent of anthracnose fruit rot, also known as black spot (Mertely and Legard, 2004). The worldwide distribution of the three species may vary. In the USA, C. gloeosporioides and C. fragariae have been described as causing severe losses, mainly in the southeast region (Florida), while Colletotrichum acutatum is the major species present in the south-west (California). However, in Europe, anthracnose is most often caused by Colletotrichum acutatum. The strawberry anthracnose symptoms produced are similar and occur most commonly on runners, flowers and fruits. In the fruit, the fungus causes circular lesions, which are firm and sunken and become black spots on mature fruit (Garrido et al., 2008). Within the crown tissue, Colletotrichum spp. cause red-brown discoloration and necrosis of the fruits (Garrido et al., 2008; Smith, 2008), or even the wilting of infected plants during periods of moisture stress, such as early afternoon in the summer. Under environmental conditions that favour infection, this process may continue for several days until the crown infection is extensive and causes the entire plant to wilt and die. To colonize the host, these fungi develop many specialized infection structures, including germ tubes, appresoria, intracellular hyphae and secondary necrotrophic hyphae. The infection begins when the conidia adhere to plant surfaces, produce germ tubes and then continue forming appresoria, which penetrate the cuticle directly (Curry et al., 2002). The pathogen grows beneath the cuticle by forming a subcuticular intramural network of hyphae before spreading throughout the tissue. 163 In the case of C. gloeosporioides, the process continues with the formation of secondary necrotrophic hyphae, which kill plant cells by using cellular debris as nutrients (Bailey et al., 1992). Control of anthracnose can be difficult, as few available fungicides are effective against anthracnose of strawberry; once an epidemic of anthracnose fruit rot begins in a susceptible cultivar, it is nearly impossible to control (Maas, 2004). The prospects for developing cultivars resistant to anthracnose are relatively good. Although the genetics of anthracnose resistance are complicated, gains from recurrent selection have been possible because of the high broad-sense heritability estimates for resistance (Maas, 2004). Currently, only a few resistant cultivars are available. In the work performed by Seijo et al. (2008), the cultivars ‘Sweet Charlie’, ‘Ruby Gem’, ‘Florida Elyana’ and ‘Florida Radiance’ proved to be the most resistant; ‘Strawberry Festival’ and advanced selection 99-117 were intermediate in susceptibility; and ‘Albion’, ‘Camarosa’, ‘Camino Real’, ‘Ventana’, ‘Candonga’ and ‘Treasure’ were susceptible/highly susceptible. Transformation of strawberry plants using Agrobacterium-mediated transformation has shown that expression of a β-1,3glucanase gene, isolated from the antagonist soil fungus Trichoderma harzianum, in strawberry enhanced anthracnose resistance (Mercado et al., 2007). 10.2.3. Fusarium oxysporium Within the genus Fusarium, two species have been described, Fusarium oxysporium and Fusarium sambucinum (Table 10.1), that are capable of causing significant damage to strawberry plants. Fusarium oxysporium is the main cause of the disease called Fusarium wilt, while Fusarium sambucinum is responsible for the disease called fruit blotch and the root rots or storage rot. However, it is Fusarium oxysporum that is the major cause of economic loss in the cultivation of strawberries, and therefore there have been a greater number of studies about its biological and morphological description, mode of infection and pathogen control. ©CAB International 2016 – for Amjad M.Husaini 164 C. Garrido et al. The ascomycete Fusarium oxysporium causes disease on many economically important crop species. This cosmopolitan soilborne fungus is considered a normal constituent of the fungal rhizosphere community of plants (Fravel et al., 2003). Propagules of the pathogen can survive for long periods in soil organic matter or the rhizosphere of many plant species (Fravel et al., 2003), which may serve as the primary inoculum for other susceptible hosts. The fungus penetrates strawberry plants through the roots, colonizes the root cortex and then grows into the xylem, disrupting water transport throughout the plant. This leads to the typical symptoms of reddish-brown discoloration on the crown, drying and early senescence of mature leaves, followed by stunting and wilting of the entire plant. In addition, transplants are infected through runners from infected mother plants (Nam et al., 2009). Wilt-inducing isolates of Fusarium oxysporium are host-specific fungal pathogens and have been divided into more than 120 different formae speciales (f. sp.) based on their host specificity (Fravel et al., 2003). Among these, Fusarium oxysporium f. sp. fragariae is the specific pathogen responsible for the wilt of strawberry. Fusarium oxysporium f. sp. fragariae has been reported from strawberry-producing regions throughout the world, including Australia, Korea, China, Spain and the USA (Fang et al., 2012). Traditionally, management of Fusarium wilt on strawberry plants has been mainly through chemical soil fumigation; however, the most cost-effective and environmentally sustainable strategy is the use of resistant cultivars. The successful development and deployment of resistant cultivars requires an understanding of the defence responses of strawberry against Fusarium oxysporium f. sp. fragariae, which remain poorly understood (Fang et al., 2013). Major gene resistance has provided effective control of Fusarium wilt in many crops, although the durability of resistance has been variable, with the emergence of new pathogenic resistant races (Islas, 2012). Fang et al. (2012) has reported that the strawberry cultivar ‘Camarosa’ is the most susceptible cultivar to Fusarium oxysporium f. sp. fragariae, while ‘Festival’ is the most resistant cultivar. The development of strawberry cultivars resistant to Fusarium wilt is currently one main focus area in the University of California strawberry breeding programme (Islas, 2012). 10.2.4. Phytophthora spp. The oomycete genus Phytophthora comprises fungus-like organisms; it includes more than 80 species and harbours a group of important plant pathogens, capable of causing considerable economic losses to food crops and ornamentals. Of these, at least eight species (Table 10.1) have been isolated from the roots, crowns, stolons and fruits of affected strawberry plants in different regions of the world (Abad et al., 2008). Species of Phytophthora associated with strawberry include Phytophthora bisheria, Phytophthora cactorum, Phytophthora citricola, Phytophthora citrophthora, Phytophthora cryptogea, Phytophthora fragariae var. fragariae, Phytophthora megasperma and Phytophthora nicotianae. The species Phytophthora cactorum and Phytophthora fragariae var. fragariae are the most important pathogens and occur in almost all countries where strawberry is cultivated. Phytophthora cactorum has been reported as the causal agent of more than 200 plant species diseases from over 60 different families (Eikemo et al., 2004). In strawberry, it causes leather rot of the fruit, stem and crown, and root rot (Abad et al., 2008). These diseases have been described in the USA and other temperate to subtropical regions. In general, Phytophthora cactorum has a wide host range; however, not all strains are able to infect all host species. Phytophthora cactorum isolated from strawberry crowns has been shown to be genetically very uniform and has been suggested to originate from a single clone, at least within the EU (Chen et al., 2011). The analysis of microsatellites of Phytophthora cactorum from strawberry showed that leather rot of strawberry fruit and crown rot are not caused by genetically different strains of ©CAB International 2016 – for Amjad M.Husaini Fungal Diseases of Strawberry the species (Hantula et al., 2000). Eikemo et al. (2004) discovered than none of the Phytophthora cactorum isolates from other hosts could cause crown rot symptoms in strawberry. Therefore, the causal agent of crown rot is often referred to as a distinct pathotype of Phytophthora cactorum, as it cannot be distinguished morphologically from leather rot (Hantula et al., 2000). Infection usually occurs during warm periods with prolonged wetness. Symptoms typically develop during early to mid-summer. The youngest leaves turn bluish green and often wilt suddenly; wilting quickly spreads to the entire plant, which collapses and dies, typically within a few days. Red stele root, caused by Phytophthora fragariae var fragariae, was first observed in Scotland and is generally most severe in areas with cool and moist climates. There are no known natural hosts other than strawberry and loganberry, although artificial infection of other hosts has been possible. Symptoms usually appear on the upper parts of plants that come under stress in late spring or early summer, especially in low-lying wet areas. They may die just before fruiting or may produce a few small fruits. Younger leaves can have a blue-green coloration, while older ones turn yellow or red. In the root, there is a reddish discoloration of the steles, which occurs when the soil is cool, and the root later starts to rot from the tip upwards. Plants with severe root rot are often stunted and may wilt in hot weather. The control of diseases caused by Phytophthora spp. is often difficult due to the release into the soil of resistant perennating structures, oospores and/or chlamydospores. Traditionally, Phytophthora spp. have been controlled by pre-plant soil fumigation or applying fungicides or mixtures of the two. Soil fumigation may reduce the inoculum in the soil but may not eradicate the fungus, and the use of fungicides has led to development of resistant strains (Maas, 2004). The use of resistant cultivars is by far the most reliable way to avoid root rot and crown rot problems. Many cultivars are available in North America and Europe that are resistant to this disease. In the case of 165 cultivars resistant to Phytophthora fragariae var fragariae, there exists a race-specific resistance to the simpler races. Thus, most strawberry breeding programmes have concentrated on incorporating resistance against multiple races and have extended the range of useful cultivars that can be planted in sites infested with multiple races of this pathogen. This has been complicated in the past because the nature of genetic resistance was largely unknown and crossing two red stele-resistant parents often gave unpredictable results. However, recent studies have shown that the control of resistance and pathogenicity involves at least five genes for resistance (Maas, 2004). 10.2.5. Verticillium spp. Verticillium is a genus of fungi belonging to the phylum Ascomycota, and causes wilt of over 300 plant species. Two Verticillium spp., Verticillium dahliae and Verticillium albo-atrum (Table 10.1) cause enormous economic losses in the cultivation of strawberries because they cause vascular diseases in the plants (Bhat and Subbarao, 1999). These soilborne plant pathogens are distributed throughout the world. Verticillium fungi overwinter in soil and plant debris as dormant mycelium or microsclerotia. When the conditions are favourable, the microsclerotia germinate and the hyphae may penetrate the root hairs directly, but infection is aided by breaks or wounds in the rootlets. Once inside the root, the fungus invades and destroys the xylem, resulting in reduced water uptake by the plant, causing the plants to wilt and wither. The symptoms produced by this pathogen vary depending on the susceptibility of the cultivar and cannot easily be differentiated from those for red stele or black root rot. The initial symptoms appear rapidly, when a sudden onset of high temperatures, high light intensity or drought interrupt mild conditions. Verticillium wilt tends to be most severe in plants that are already fruiting, and symptoms may continue throughout the summer and autumn. When the infection is established, the leaves droop, wilt, turn dry and become ©CAB International 2016 – for Amjad M.Husaini 166 C. Garrido et al. reddish-brown or dark yellow at the edges and between the veins. The strawberry plants are stunted and flattened with small yellowish leaves due to lack of water. Brown-bluish black streaks or smears may appear on runners and leaf petioles. The new roots growing from the crown often become overshadowed with blackened tips and the inside of the crown may appear to have brownish stripes. Excellent long-range control of Verticillium wilt has been obtained by pre-plant soil fumigation or by the use of resistant strawberry cultivars. Several old and new cultivars are available that are moderately to highly resistant to Verticillium wilt. The transformation of strawberry cultivars using Agrobacterium-mediated transformation protocol has also been successful. Chalavi et al. (2003) carried out the transformation of ‘Joliette’ strawberry with a chitinase gene (pcht28) from Lycopersicon chilense. In growth chamber studies, they observed that the transgenic strawberry plants that expressed pcht28 had significantly higher resistance to V. dahliae. 10.2.6. Other genera As well as the above-discussed fungal pathogens, there are other types of fungi capable of causing damage to the fruit, crown and root of strawberry plants whose incidence in the field is much smaller, and therefore the economic losses in strawberry cultivation are lower. Within the genera of fungi responsible for damage to the fruit are Rhizoctonia fragariae (anther and pistil blight), Mycosphaerella fragariae (black seed disease) and Sclerotinia sclerotiorum (sclerotinia fruit rot). This group of fungi vary widely in severity of disease, and no specific control measures have been developed against them, although pre-plant soil treatment and cultivation practices, such as mulching and removal of plant debris, may help to minimize their incidence. Others, such as Gnomonia comari (stem end rot), Pestalotia longisetula (Pestalotia fruit rot), Alternaria spp. (Alternaria rot) and Cladosporium spp. (Cladosporium rot), Aspergillus niger (Aspergillus rot), and Stagonospora fragariae (Stagonospora hard rot) are of less commercial importance. Lastly, there is one group of filamentous fungi that are especially important because they cause postharvest losses to strawberry in storage. This group includes Rhizopus stolonifer and Rhizopus sexualis (Rhizopus rot); Mucor mucedo, Mucor piriformis and Mucor hiemalis (Mucor fruit rot); Penicillium cyclopium, Penicillium frequentans, Penicillium expansum and Penicillium purpugenum (Penicillium fruit rot). Additionally, some yeasts also cause damage at postharvest, such as Saccharomyces cerevisiae, Saccharomyces kluyveri, Pichia membranifaciens, Pichia subpelliculosa, Zygosaccharomyces bailii and Zygosaccharomyces florentinus (Douglas Gubler and Converse, 1993). The importance of these pathogens in causing postharvest rot has been substantially reduced by modern storage and shipping methods (Maas, 1998). The crowns and roots of strawberry plants may be also attacked by other fungi (Table 10.1), with less serious commercial consequences. These include Armillaria mellea (Armillaria root and crown rot), Coniothyrium fragariae and Coniothyrium fuckelli (black root rot), Cylindrocarpon destructans (root rot), Dematophora necatrix (Dematophora root and crown rot), Hainesia lythri (black root rot), Idriella lunata (Idriella root rot), Macrophomina phaseolina (Macrophomina root rot), Phoma lycopersici (leaf stalk rot) and Phoma terrestris (grey sterile fungus root rot), Rosellinia necatrix (white root rot) and Sclerotinia sclerotiorum (Sclerotinia crown rot) (Douglas Gubler and Converse, 1993; Agrios, 2011) The genus Pythium spp., especially the species Pythium ultimum, is the most widespread strawberry root pathogen favoured by cool climates. This species has been shown to be a major cause of black root rot disease. Pythium spp. are not only strawberry pathogens; they can attack many other crops, both annual and perennial. In strawberry, Pythium spp. destroy juvenile root tissue, such as feeder rootlets. Control of Pythium spp. is effective with the application of chloropicrin mixtures. ©CAB International 2016 – for Amjad M.Husaini Fungal Diseases of Strawberry Two other species that are able to kill structural roots as well as feeder rootlets of strawberries are Rhizoctonia solani and Rhizoctonia fragariae. Lesions on young roots are reddish-brown at first, and darken with age. The infected crowns show internal brown discoloration of basal tissues and frequently collapse and die. Traditionally, these diseases have been controlled by fumigating the soil prior to planting (Garrido et al., 2010). The leaf of the plant is also an organ that can be affected by fungal pathogens. There are at least 19 species that cause leaf damage, some only occasionally, depending on the susceptibility of cultivar and environmental conditions (Table 10.1). When enough leaf tissue has been destroyed by disease, the plant is weakened, and in such cases, the plant is more subject to winter injury. Additionally, the pathogens that cause these diseases on leaves can infect the berries, causing quality problems or even loss of fruits. Alternaria alternata (Alternaria black leaf spot), Colletotrichum acutatum, Colletotrichum gloeosporioides and Colletotrichum fragariae (anthracnose leaf spot and irregular leaf spot); Diplocarpon earlianum (leaf scorch), Mycosphaerella fragariae (leaf spot), Phomopsis obscurans (Phomopsis leaf blight), Rhizoctonia solani (Rhizoctonia leaf blight) and Sphaerotheca macularis (powdery mildew) are fungi causing lesions on the leaves. These diseases occur on strawberry plants in all areas of the world, from temperate climates to subtropical and tropical regions. The major effect of these pathogens is the progressive destruction of the foliage, which may weaken plants and reduce yields. Some pathogens cause very distinctive leaf symptoms; for example, Sphaerotecha macularis forms white patches of mycelium on the abaxial surface of the leaf. Other species cause similar, even identical symptoms, from circular spots with grey centres and dark margins to irregular purplish red or brown areas with dark reddish purple margins. It is usual in the literature to find incorrect identifications, and many citations confirm that symptoms are often confused among pathogens; for example, leaf scorch, caused by 167 Diplocarpon earlianum is often confused with the symptoms of leaf spot caused by Mycosphaerella fragariae; and the symptoms produced by the latter are also wrongly identified as Phomopsis leaf blight caused by Phomopsis obscurans (Maas, 1998). Leaves can be also attacked by fungi other than those discussed above, but their commercial importance is minor since they are limited to a few particular regions or countries and they occur only occasionally in strawberry. These other minor diseases of leaves include Gnominia comari (leaf blotch); Mycophaerella louisianae (purple leaf spot); Septoria fragariae, S. aciculosa and S. fragariaecola (septoria leaf spots); Macrophomina phaseolina (macrophomina leaf blight); Cercospora fragariae and C. vexans (cercospora leaf spots); Sclerotium rolfsii (sclerotium rot); and Phoma lycopersici (leaf stalk rot) (Maas, 1998). 10.3. Diagnosis Methods and Field Monitoring of Strawberry Diseases 10.3.1. Molecular methods applied to phytopathogenic fungi Phytopathogenic fungi are able to infect any tissue at any stage of strawberry plant growth. These fungi show complex life cycles, including both sexual and asexual reproduction stages (Agrios, 2011). The biological variability of pathogenic races gives them the advantage of adaptation in climatologically different environments, ranging from dry and desert zones to wet and hot regions. This ability to adapt allows them to cause disease in almost all Fragaria × ananassa cultivars. The development of molecular methods has led to the generation of much information about these diseases (Garrido et al., 2009b). Traditionally, identification and characterization of fungi were focused on classical microbiological studies, but molecular methods have added to our understanding of these microorganisms. Molecular methods allow a more accurate characterization and differentiation of phytopathogenic ©CAB International 2016 – for Amjad M.Husaini 168 C. Garrido et al. fungi. These methods include iso-enzyme comparisons, restriction fragment length polymorphism (RFLP) analyses of mitochondrial DNA, AT-rich analyses, random amplified polymorphic DNA (RAPD) analyses, genusand species-specific polymerase chain reaction (PCR) methods and enzyme-linked immunosorbent assays (ELISAs) (Garrido et al., 2009a, 2012). In recent years, many changes in the cultural practices have taken place in the cultivation of crops in general, and strawberry in particular. The use of toxic chemical compounds such as methyl bromide to control and prevent the appearance of pathogens has been progressively replaced by new biological, cultural and physical methods. These methods have led to the discovery of alternative approaches to disease control. They have reduced the massive fumigant use for soil and plant treatment, replacing them with biocontrol agents (microorganisms), biofumigants and alternative non-persistent chemical compounds of biological origin. 10.3.2. Past and present diagnosis methods The first step in implementing appropriate strategies for disease management and appropriate control measures requires the unambiguous identification of the organism(s) responsible for the disease of the strawberry crop (Garrido et al., 2011). Because many fungal pathogens of strawberry produce similar symptoms, it is important to be able to distinguish between different species. After accurate identification of the pathogen, it is essential to design a correct programme for its management. Many pathogens are subjected to special regulation through quarantine programmes agreed among producer countries. Therefore, pathogen identification is crucial to all aspects of fungal diagnostics and epidemiology in the field of plant pathology, and also in medical science, environmental studies and biological control (McCartney et al., 2003; Atkins and Clark, 2004). The best way forward for pathogen identification is to use faster methods, making it possible to detect diseases even before symptoms appear (Debode et al., 2009; Garrido et al., 2009a). Fungal pathogens cause diseases in strawberry using different reproductive structures, including asexual and sexual life cycles. Morphological characterization of the structures developed by fungal pathogens has been the basis used by researchers for identifying organisms to the genus/species level, and for classifying these pathogens into families, orders and classes. This classical method has been used for the detection and identification of fungal pathogens, including visual interpretation of plant symptoms, characterization of fungal structures and biochemical/chemical analyses. These studies have contributed much information, increasing our biological knowledge of these fungal species, but they have many limitations pertaining to accuracy and reliability in the detection/diagnosis. They are often time-consuming and laborious methods, and the organisms themselves must be capable of being cultured in the laboratory. This fact is a significant handicap, because less than 1% of the microorganisms present in an environmental sample can be cultured (Lievens et al., 2005). Such classical analyses also require experienced and skilled laboratory staff, who need to have extensive knowledge of taxonomy (McCartney et al., 2003). In the past two decades, new methodologies based on molecular and immunological strategies have been used including among others: RFLP analyses of mitochondrial DNA (Sreenivasaprasad et al., 1992; Garrido et al., 2008), amplified fragment length polymorphism (AFLP), AT-rich analyses (Freeman et al., 2000), RAPD (Whitelaw-Weckert et al., 2007), genus- and species-specific PCR analysis (Mills et al., 1992; Sreenivasaprasad et al., 1992; MartinezCulebras et al., 2003; Garrido et al., 2008; Capote et al. 2012), real-time PCR studies (Garrido et al., 2009a) and ELISA assays (Hughes et al., 1997). These techniques minimize the time to diagnosis and increase accuracy in identification of the microorganisms (Atkins and Clark, 2004; Capote et al. 2012). ©CAB International 2016 – for Amjad M.Husaini Fungal Diseases of Strawberry 10.3.3. PCR alternatives applied to fungal diagnosis PCR is the most important and sensitive technique presently available for the detection of plant pathogens. Advances in PCR technology have opened up alternative approaches to the detection and identification of strawberry fungal pathogens, even onsite alternatives under field conditions, away from the laboratory, are now available, providing results in a short time (Capote et al. 2012). The evolution of PCR towards realtime PCR allows faster and more accurate detection and quantification of plant pathogens in an automated reaction. The advantages of PCR techniques are the high sensitivity, high specificity and reliability. Moreover, it is not necessary to isolate the pathogen from the infected material, reducing the diagnosis time from weeks to hours, and allowing the detection and identification of non-culturable pathogens. This characteristic has been especially useful in the analysis of symptomless plants. However, the frequent presence of PCR inhibitors in plant tissues or soil can reduce considerably the sensitivity of the reactions and may even result in false-negative results. The optimization of an experimental PCR set-up focuses on three fundamental steps: (i) extraction of total community DNA/ RNA from the environmental sample; (ii) selection of a specific target region of the DNA/ RNA to identify the fungus; (iii) identification of the presence of the target DNA/RNA region in the sample (Atkins and Clark, 2004; Capote et al. 2012). In recent years, many research studies have been published reporting improvements to each of the fundamental steps described above, and working on some of the most serious fungal pathogens of strawberry, such as B. cinerea (Suarez et al., 2005), Colletotrichum acutatum (Debode et al., 2009; Garrido et al., 2009a), Colletotrichum gloeosporioides, Colletotrichum spp. (Garrido et al., 2009a), Fusarium oxysporum (Lievens et al., 2003), Verticillium albo-atrum (Larsen et al., 2007) and Verticillium dahliae (Atallah et al., 2007). The first step in PCR diagnostic analysis consists of collection of the samples 169 from the plant material. This can be symptomatic or non-symptomatic tissue. An efficient and reliable DNA extraction method is then used. Commercial kits are now available for the extraction of fungal DNA from environmental samples such as plant tissue or soil, supplied by several companies (e.g. Dynabeads® DNA Direct from ThermoFisher Scientific; Soil DNA Isolation kit from Mo Bio Laboratories), but these kits can be costly and are not always totally reliable with respect to co-extraction of PCR inhibitors. Garrido et al. (2009a) optimized a DNA extraction protocol that can be used for samples of strawberry plant material directly, or from fungal colonies removed from an agar plate. This method uses sample material physically ground using a grinding machine, in the presence of CTAB (cetyl trimethyl ammonium bromide) lysis buffer. Garrido et al. (2009a) demonstrated that this method is very reliable for extracting DNA from any strawberry plant material. The sensitivity and accuracy of PCR protocols depends mainly on the instrumentation and technique used (i.e. conventional PCR versus real-time PCR), but in a high proportion of cases, this sensitivity depends on the quality of the total community DNA/RNA extracted from the environmental samples. One disadvantage of PCR-based methods is the inability to discriminate viable from non-viable fungi or fungal structures. The detection of mRNA by reverse transcription (RT)-PCR is considered an accurate indicator of cell viability (Sheridan et al., 1998). In RT-PCR, the RNA is reverse transcribed using the enzyme reverse transcriptase. The resulting cDNA is then amplified using a PCR-based method. The most frequent application of this technique in phytopathology is the analysis of plant and fungal gene expression during disease development (Yang et al., 2010). Many PCR variations have been developed to improve the sensitivity, specificity, speed and throughput, and to allow the quantification of the fungal pathogen in the plant and the environment. A second disadvantage is the selection of the target DNA/RNA to amplify. Known ©CAB International 2016 – for Amjad M.Husaini 170 C. Garrido et al. conserved genes with enough sequence variation are selected and a PCR diagnostic assay can then be designed to perform phylogenetic analysis. The ribosomal RNA (rRNA) genes are the regions that were used by Debode et al. (2009) and Garrido et al. (2009a) to develop protocols for identification of members of the genus Colletotrichum, and to distinguish between the three species causing anthracnose in strawberry: Colletotrichum acutatum, Colletotrichum gloeosporioides and Colletotrichum fragariae. These regions are the most commonly used DNA regions targeted to design primers for PCR-based identification and detection of fungal plant pathogens, because of the highly variable sequences of the internal transcribed spacers, ITS1 and ITS2, which separate the 18S/5.8S and 5.8S/28S rRNA genes, respectively (Garrido et al., 2009a). As recent examples, PCR methods for identification of Sclerotium rolfsii (Jeeva et al., 2010) and Colletotrichum capsici (Torres-Calzada et al., 2011) were developed based on specific sequences of the ITS region. Another highly variable region of the rRNA genes is the intergenic spacer region, which separates the 28S/18S rRNA genes (Suarez et al., 2005), but these regions have been less used than ITSs. Other regions are becoming more widely studied, for example, sequences of the β-tubulin gene (Suarez et al., 2005; Atallah et al., 2007; Debode et al., 2009), translation elongation factor 1α (Geiser et al., 2004; Knutsen et al., 2004; Kristensen et al., 2005), calmodulin (Mulè et al., 2004), avirulence genes (Lievens et al., 2007, 2008) and mitochondrial genes such as the multicopy cox I and cox II and their intergenic region (Martin and Tooley, 2003; Seifert et al., 2007; Nguyen and Seifert, 2008). The third step in effective screening protocol is the detection step. Many variants of conventional PCR such as nested PCR and co-operational amplification (coPCR), multiplex PCR, PCR-ELISA, RT-PCR, PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time-PCR have been developed to improve the sensitivity, specificity, speed and performance, and to permit quantification of the plant pathogenic fungus. Co-PCR is a method that enhances sensitivity and minimizes contamination risks. In co-PCR, a single reaction containing four primers, one pair internal to the other, enhances the production of the longest fragment by the co-operational action of all amplicons (Olmos et al., 2002). Co-PCR is usually coupled with dot blot hybridization by using a specific probe to enhance the specificity of the detection and provides a sensitivity level similar to nested PCR. Nested PCR consists of two consecutive rounds of amplification in which two external primers amplify a large amplicon, which is then used as a target for a second round of amplification using two internal primers (Porter-Jordan et al., 1990). This method has been widely used for detection and/or further characterization of numerous fungi (Hong et al., 2010; Meng and Wang, 2010; Qin et al., 2011; Wu et al., 2011). In both nested and co-PCR, the use of external primers can be used for generic amplification and the internal primers for further and more specific characterization of the amplified product at the species or strain level. Real-time PCR (also known as quantitative PCR or qPCR) is currently considered the best method for detection of plant pathogens. There are many advantages of real-time PCR over conventional PCR, including the fact that this system does not require the use of post-PCR processing (e.g. electrophoresis, hybridization), avoiding the risk of carryover contamination and reducing assay labour and material costs. Real-time PCR is more sensitive, more accurate and less time consuming than conventional end-point qPCR (Lievens et al., 2005). It allows monitoring of the reaction during the amplification process by use of a fluorescent signal that increases proportionally with the number of amplicons generated and with the number of targets present in the sample. For the fluorescent signal, different chemistries can be used, such as TaqMan®, SYBR® Green I, molecular beacons, Scorpions® or a new approach, developed by Invitrogen, called LUX. The TaqMan® system consists of a fluorogenic probe specific to the DNA target, which anneals to the target between the PCR primers. ©CAB International 2016 – for Amjad M.Husaini Fungal Diseases of Strawberry The TaqMan® system tends to be the most sensitive of the methods but the new approach, LUX, is achieving the same level of sensitivity while further simplifying the techniques, as well as reducing the cost. This new system uses two primers, one of which has a hairpin-loop structure with a fluorophore (Suarez et al., 2005). Although this method is replacing TaqMan® probes in real-time PCR technology, with the objective of providing cheaper, reliable methods with the specificity of TaqMan® probes without some of the constraints, to date TaqMan® technology continues to be used in the majority of research projects working with strawberry fungal pathogens. SYBR® Green I is a fluorescence intercalating dye with a high affinity for double-stranded DNA. The overall fluorescent signal from a reaction is proportional to the amount of double-stranded DNA present in the sample. Molecular beacons (Tyagi and Kramer, 1996; Capote et al., 2012) are specific oligonucleotide probes (15–40-mers) flanked by two complementary 5–7-mer arm sequences, with a fluorescent dye covalently attached to the 5′ end and a quencher dye at the 3′ end. When the probe hybridizes to the target sequence, this allows the emission of a fluorescent signal. Molecular beacons have allowed real-time specific quantification of Fusarium equiseti (Maciá-Vicente et al., 2009). Scorpions® are bifunctional molecules in which an upstream hairpin probe is covalently linked to a downstream primer sequence (Whitcombe et al., 1999). The hairpin probe contains a fluorophore at the 5′ end and a quencher at the 3′ end. The loop portion of the scorpion probe is complementary to the target sequence. The level of specificity and sensitivity of real-time PCR-based methods with some of the main strawberry fungal pathogens reported in several publications vouch for the results offered by this technology. Suarez et al. (2005) designed three TaqMan® probe/primer sets based on the ribosomal intergenic spacer, the β-tubulin gene and the sequencecharacterized amplified region (SCAR) marker of B. cinerea published by Rigotti et al. (2002). For the detection of Colletotrichum spp. and for monitoring strawberry anthracnose 171 using real-time PCR, new protocols were published by Debode et al. (2009) and Garrido et al. (2009a). Both groups mainly used the ITS regions to design the sets of probe/ primers. Garrido et al. (2009a) tested the specificity of all assays using DNA from isolates of six species of Colletotrichum and from DNA of another nine fungal species commonly found associated with strawberry material. Additionally, they checked that samples did not contain PCR inhibitors co-extracted during the DNA extractions using one universal pair of primers for the 5.8S rRNA gene by PCR SYBR® Green I amplifications. These assays were highly specific for Colletotrichum spp., Colletotrichum acutatum and Colletotrichum gloeosporioides, and no cross-reactions were observed with either related plant pathogens or healthy strawberry plant material. The real-time PCR assay detected the equivalent of 7.2 conidia per plant inoculated with a serial dilution of Colletotrichum acutatum spores, demonstrating the high degree of sensitivity (Garrido et al., 2009a). The sensitivity of the new real-time PCR assays was compared with that of previously published conventional PCR assays; they were confirmed to be 100 times more sensitive than the latter. Bilodeau et al. (2011) developed a new assay for rapid detection and quantification of V. dahliae, responsible for strawberry wilt, which can cause significant crop loss. To provide a faster means for estimating pathogen populations, a multiplexed TaqMan® realtime PCR assay based on the rRNA gene intergenic spacer was developed for V. dahliae. Variation in copy number of the rRNA gene was also evaluated among isolates by SYBR® Green I real-time PCR amplification of the V. dahlia-specific amplicon, compared with amplification of several single-copy genes, and was estimated to range from approximately 24 to 73 copies per haploid genome, which translated into possible differences in results among isolates of around 1.8 cycle thresholds. For the assay, they used an internal control for valuation of inhibition due to the presence of PCR inhibitors in DNA extracted from soil samples. This method provides an accurate and ©CAB International 2016 – for Amjad M.Husaini 172 C. Garrido et al. rapid means for quantification of V. dahliae over a wide range of inoculum densities. The species-specific marker for V. dahliae described can be used for pathogen identification and quantification in plant tissue, but in this case, another internal control must be used that will allow comparison of the quantity of pathogen relative to the plant DNA. Likewise, due to the unique sequence of the internal control and the ease with which it can be modified to be amplified with other primers, the internal control from the V. dahliae assay could also be used in other molecular quantification assays. A good correlation was observed in regression analysis (R2 = 0.96) between real-time PCR results and inoculum densities determined by soil plating in a range of field soils with pathogen densities as low as one to two microsclerotia g–1 soil. Although real-time PCR is currently considered the best standard method for detection of plant pathogens, other assay formats are applied to plant pathogens, such as multiplex PCR, which is based on the use of several PCR primer pairs in the same reaction, allowing the simultaneous and sensitive detection of different DNA targets, reducing both time and cost. This method is useful in plant pathology, as plants are usually infected by more than one pathogen. This needs an accurate and careful design of primers, and optimization of their relative concentrations is required to obtain equilibrate detection of all target fungi. Multiplex PCR has been used for differentiating two pathotypes of Verticilliun albo-atrum infecting hop (Radišek et al., 2004). Another multiplexing method that allows simultaneous detection and identification of multiple oomycetes and fungi in complex plant or environmental samples is the use of the ligation detection system using padlock probes. Padlock probes are long oligonucleotide probes containing asymmetric target-complementary regions at their 5′ and 3′ ends and also incorporate a desthiobiotin moiety for specific capture and release, an internal endonuclease IV cleavage site for linearization and a unique sequence identifier for standardized microarray hybridization. Padlock probes have been used for the simultaneous detection of Phytophthora cactorum, Phytophthora nicotianae, Pythium ultimum, Pythium aphanidermatum, Pythium undulatum, Rhizoctonia solani, Fusarium oxysporum f. sp. radicis-lycopersici, Fusarium solani, Myrothecium roridum, Myrothecium verrucaria, V. dahliae and V. alboatrum in samples collected from horticultural water circulation systems in a single assay (van Doorn et al., 2009). 10.3.4. Other molecular techniques used to study fungal pathogens A combination of PCR and ELISA has been used for the detection of several species of Phytophthora and Pythium. This method is as sensitive as nested PCR and can easily be automated, and hence is very suitable for routine diagnostic purposes. It is based on the use of forward and reverse primers carrying biotin and an antigenic group at their 5′ ends. PCR-amplified DNA can be immobilized microtiter plates (e.g. on avidin- or streptavidin-coated plates) via the biotin moiety of the forward primer and then can be quantified by an ELISA specific for the antigenic group of the reverse primer. A PCR-DGGE detection tool based on the amplification of the ITS region has been applied recently to detect multiple species of Phytophthora from plant material and environmental samples (Lilja et al., 2008; Capote et al., 2012). In PCR-DGGE, target DNA from plant or environmental samples is first amplified by PCR and is then subjected to denaturing electrophoresis. Sequence variants of particular fragments migrate to different positions in the denaturing gradient gel, allowing very sensitive detection of polymorphisms in DNA sequences. The bands obtained in the gel can be extracted, cloned or reamplified, and sequenced for identification. These techniques are suitable for the identification of novel or unknown organisms, and the most abundant species can readily be detected. However, this method has some problems because it is time consuming, is poorly reproducible and the analysis of complex communities of microorganisms may be ©CAB International 2016 – for Amjad M.Husaini Fungal Diseases of Strawberry difficult due to the large number of bands obtained. Fingerprinting analyses have also been useful for identifying specific sequences used for the detection of fungi at very low taxonomic level, and even to differentiate strains of the same species with different host ranges, virulence, compatibility groups or mating types. The most prominent of these techniques are RFLP, RAPD, AFLP and the use of microsatellites. RFLP involves restriction enzyme digestion of the pathogen DNA, followed by separation of the fragments by electrophoresis in agarose or polyacrylamide gels to detect differences in the size of DNA fragments (Capote et al., 2012). These differences are used to distinguish fungal species. This early technique has been progressively supplanted by PCR-RFLP, which combines the amplification of a target region with the further digestion of the PCR products. PCRRFLP analysis of the ITS region demonstrated the presence of different anastomosis groups within isolates of Rhizoctonia solani (Pannecoucque and Höfte, 2009; Capote et al., 2012); PCR primers specific to members of the genus Phytophthora were used to amplify and further digest the resulting amplicons, yielding a specific restriction pattern of 27 different Phytophthora spp. (Drenth et al., 2006). In some cases, when data obtained using specific sequences from databases are inadequate because a specific primer cannot be designed, screening of arbitrary regions of the genome is often the next step. This strategy consists of an initial RAPD screening and subsequent analyses of the products with the object of developing a SCAR marker. This protocol was used by Larsen et al. (2007) to develop SCAR markers for Verticillium albo-atrum. Rigotti et al. (2002), studying B. cinerea isolates, identified SCAR markers for the specific identification of this pathogen in Fragaria × ananassa. Three years later, Suarez et al. (2005) compared their primers designed in the intergenic spacer regions and showed that this new assay was more sensitive than all the previous assays for B. cinerea. RAPD assays have been used to analyse the genetic diversity 173 among different species and races of Fusarium spp. (Drenth et al., 2006; Lievens et al., 2007). SCAR primers have been used to distinguish among several Fusarium oxysporum f. sp. (Lievens et al., 2008). The results obtained from RAPD profiles are easy to interpret. This is a rapid and inexpensive technique, but high-quality DNA is needed, although only in low quantity (Chandra et al., 2011). These methods have a problem with many basidiomycetes and oomycetes that are heterokaryons, diploids or polyploids (Fourie et al., 2011) because RAPD is a dominant marker and so cannot measure the genetic diversity affected by the number of alleles at a locus, or differentiate homozygote and heterozygote individuals. AFLP technology has the capability to amplify between 50 and 100 fragments at one time and to detect various polymorphisms in different genomic regions simultaneously. This technique consists of the use of restriction enzymes to digest total genomic DNA, followed by ligation of restriction half-site specific adaptors to all restriction fragments. A selective amplification of these restriction fragments is performed with PCR primers that have within their 3′ end the corresponding adaptor sequence and selective bases. Depending on the primers used and the reaction conditions, amplification of fungal genomes produces genetic polymorphisms specific to the genus, species or strain (Liu et al., 2009; Capote et al., 2012). This technique is reproducible and highly sensitive, but a disadvantage is that it requires a larger quantity of DNA. AFLP has been used to differentiate fungal isolates at several taxonomic levels and to separate non-pathogenic strains of Fusarium oxysporum (Stewart et al., 2006). AFLP profiles have also been widely used for the phylogenetic analysis of Fusarium oxysporum complexes (Baayen et al., 2000; Groenewald et al., 2006; Fourie et al., 2011; Capote et al., 2012). Microsatellites, also known as simple sequence repeats or short tandem repeats, can differ in repeat number among individuals, and their distribution in the genome is almost random. They have been used for studies of the genetic diversity of plant ©CAB International 2016 – for Amjad M.Husaini 174 C. Garrido et al. pathogenic fungi within species, for example in Sclerotinia sclerotiorum (Winton and Hansen, 2001). Recently, they have also been utilized for the development of a system for detection and differentiation of phytopathogenic fungi using DNA hybridization technology. Currently, this is one of the most suitable techniques for detecting and quantifying multiple pathogens present in a sample (plant, soil or water) in a single assay. A DNA array is a collection of speciesspecific oligonucleotides or cDNAs (known as probes) immobilized on a solid support that is subjected to hybridization with a labelled target DNA. This technology has been applied to the detection of oomycete plant pathogens using specific oligonucleotides designed on the ITS region (Anderson et al., 2006; Izzo and Mazzola, 2009). Lievens et al. (2005) developed a DNA array for the specific detection and identification, within 24 h, of the strawberry fungal pathogen Fusarium oxysporium and the quarantine pathogens V. albo-atrum and V. dahliae. This assay has proved to be highly sensitive, detecting 2.5 pg of DNA for V. dahliae, 0.35 pg of DNA for V. albo-atrum and 0.5 pg of DNA for F. oxysporium. Using a cox I high-density oligonucleotide microarray, Chen et al. (2009) could identify Penicillium spp. Moreover, Lievens et al. (2007) could detect and differentiate F. oxysporum f. sp. cucumerinum and F. oxysporum f. sp. radicis-cucumerinum pathogens using a DNA array containing genus-, species- and f. sp.-specific oligonucleotides. This arraybased detection procedure for plant pathogens has been shown to be relatively costeffective, and may lead to a comprehensive pathogen assessment method for detecting and quantifying all known pathogens (fungi, bacteria and nematodes, as well as viruses) (Capote et al., 2012). In 2011, Huang et al. (2011) demonstrated the usefulness of the heteroduplex mobility assay for phylogenetic analysis and for the discrimination of closely related Colletotrichum spp. This assay is a sequence-dependent method that has been widely used to determine sequence divergence and phylogenetic relationships of closely related organisms including fungi, viruses, phytoplasma, bacteria and protozoan parasites. It was demonstrated to detect single-base differences among sequences. For this reason, heteroduplex DNA patterns (HPs) using ITS regions were used as a tool to simplify the methodology for Colletotrichum spp. identification, as the unique HPs observed resemble a ‘barcode’ (White et al., 1990). In this study, 29 monoconidial strains of Colletotrichum spp., including 18 Colletotrichum gloeosporioides, two Colletotrichum capsici, two Colletotrichum graminicola, one Colletotrichum acutatum, one Colletotrichum musae, one Colletotrichum dematium, one Colletotrichum lindemuthianum and three other unidentified species of Colletotrichum, were used. The fragments for HP analysis were prepared by PCR amplification of the ITS regions from 29 Colletotrichum strains using primers ITS1 and ITS4 (White et al., 1990). The fragments amplified from six Colletotrichum strains, named Cg1, Cg2, Cg16, Cgr1, Cl1 and Cm1, were used as references. The barcode-like HPs obtained by cross-pairing HP analyses of these six reference strains appeared to create a model system for species/strain identification. The analysis required lowcost electrophoresis equipment and could discriminate single-based differences in sequences. The unique HPs obtained indicated a possibility of using this method as a barcode-like DNA system for discriminating economically important anthracnose fungi and possibly for the detection of newly evolved strains. HP analysis using crosspairing of ITS fragments from taxonomically defined strains was suggested as a cost-effective and reliable method for discriminating closely related species of Colletotrichum. 10.3.5. Web-based decision support systems Identificator is a new tool proposed by Pertot et al. (2012), which is based on the use of the web for visual identification of plant diseases, and, although it can be used in any crop, initially it was developed using strawberry as the crop type. The system is based on a multiaccess key of identification and ©CAB International 2016 – for Amjad M.Husaini Fungal Diseases of Strawberry specifically on the selection of pictures by the user, and can be used remotely from a desktop as well as from a smart phone or personal digital assistant. The system was developed following a simple approach: visual identification where images and/or short descriptions are used to uniquely identify diseases where possible and to suggest refining the visual identification process in cases of ambiguous identification. The identification procedure follows a fourstep process: (i) the user is requested to identify the infected parts of the plant; (ii) the user is presented with a set of images and descriptions of possible symptoms that may appear on these plant parts; (iii) the user selects all the images/descriptions that match the plant at hand; (iv) the system presents all pictures of the disease (even ones that the user did not select, possibly on other parts of the plant), together with a short description of the disease. Sometimes, a laboratory analysis is required, which may be indicated at this step by the system. There are two aspects that make Identificator unique: (i) its simplicity and (ii) its flexibility, as the system can be adapted to any crop in any language, because the information on diseases and their characteristics can be edited by the user directly into the system tables. In fact, the internet opens up new opportunities for knowledge dissemination in the form of web-based expert/ decision support systems. Scientific journals publish research from research groups across the world. This data is compiled by several associations or agencies for the scientific community, who are producing very useful databases. We will discuss three of these: the Centre for Agriculture and Biosciences International (CABI), Rothamsted Research, and the Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, for their contribution to the scientific and crop producer community. CABI (http://www.cabi.org) is a not-forprofit international organization that has a special relevance in the agriculture community because of its aim of improving people’s lives by providing information and applying scientific expertise to solve problems. Knowledge transfer from the laboratory to 175 the field is the mission of this organization, using scientific publishing, development projects, and research and microbial services. Rothamsted Research (http://www. rothamsted.ac.uk/index.php) is the longest running agricultural research station in the world, providing science advances and innovation since its foundation in 1843. This station was founded by the Biotechnology and Biological Sciences Research Council (BBSRC; http://www.bbsrc.ac.uk/home/ home.aspx), and their mission is to deliver knowledge and new practices to increase crop productivity and quality and to develop environmentally sustainable solutions for food production and bioenergy. Rothamsted Research manages a very useful database, the Pathogen–Host Interaction base (PHI-base). PHI-base is an open access internet resource that provides information on pathogenicity, virulence and effector genes from different pathogens. This information is only added to the database when it is clearly demonstrated that the research group has experimentally tested the role of each gene. The third institution is the Broad Institute of MIT and Harvard. This is a younger institution, founded less than 10 years ago, and supports many research projects and compiles useful information that includes fungal pathogens of agriculture and crop relevance. The information available in this database will be described in more detail below. The last updated version of PHI-base was released in February 2013 and includes a total of 2421 genes from 107 pathogens. Nine species of strawberry fungal pathogens are included in the updated list version: Alternaria alternata, B. cinerea, Cladosporium fulvum, Colletotrichum acutatum, Colletotrichum gloeosporioides, Fusarium oxysporum, Phytophthora cactorum, Sclerotinia sclerotiorum and V. dahliae. Between them, a total of 143 genes are described in the databases (Table 10.2), with B. cinerea and Fusarium oxysporum having the highest number of characterized genes, 66 and 38, respectively. This database is updated with the information that research groups Gene_name Phenotype of mutant PHI_ID Database Accession no. Reference Alternaria alternata AKT1 AKT2 AMT AaFUS3 aapk1 RLAP1 YAP1 AaGa1 ACTT2 AaNoxA AFT1 AFT3 AaSLT2 bcnoxA bcnoxB bcnoxR bcplc1 bcpg1 bcpme1 bcpme2 BCPGA1 bcatrA bcSAK1 Bmp3 BcatrB BMP1 BcatrB BCG1 BcBOA2 BcBOA6 BcSpl1 BcFRP1 BcCdc42 Loss of pathogenicity Loss of pathogenicity Loss of pathogenicity Loss of pathogenicity Reduced virulence Loss of pathogenicity Loss of pathogenicity Unaffected pathogenicity Reduced virulence Reduced virulence Loss of pathogenicity Loss of pathogenicity Reduced virulence Reduced virulence Reduced virulence Reduced virulence Reduced virulence Reduced virulence Reduced virulence Unaffected pathogenicity Reduced virulence Unaffected pathogenicity Reduced virulence Reduced virulence Sensitive to chemical Loss of pathogenicity Reduced virulence Reduced virulence Reduced virulence Reduced virulence Reduced virulence Unaffected pathogenicity Reduced virulence PHI:133 PHI:134 PHI:160 PHI:2303 PHI:2317 PHI:2320 PHI:2373 PHI:2414 PHI:2431 PHI:2448 PHI:508 PHI:509 PHI:2318 PHI:1023 PHI:1024 PHI:1025 PHI:1026 PHI:1027 PHI:1028 PHI:1029 PHI:103 PHI:1030 PHI:1031 PHI:1032 PHI:1160 PHI:161 PHI:202 PHI:203 PHI:2289 PHI:2290 PHI:2291 PHI:2295 PHI:2298 EMBL EMBL EMBL EMBL Uniprot Uniprot Uniprot Uniprot Uniprot Uniprot EMBL EMBL Uniprot EMBL EMBL EMBL EMBL EMBL EMBL EMBL EMBL EMBL EMBL EMBL EMBL EMBL EMBL EMBL Uniprot Uniprot Uniprot Uniprot Uniprot BAA36588 BAA36589 AAF01762 GQ414506 B0LW64 C0LD25 B9V258 D2X3B5 C9K1M7 H9AWW9 BAB69076 BAB69078 D3J127 CAP12516 CAP12517 CAP12326 AAB39564 AAC64374 CAC29255 CAD21438 AAC64374 CAA93142 CAJ85638 ABJ51957 CAB52402 AAG23132 CAB52402 CAC19871 JQ665433 B1GVX7 A6RNZ1 A6SB12 A6SSN8 Tanaka et al. (1999) Tanaka et al. (1999) Johnson et al. (2000) Lin et al. (2009) – – Lin et al. (2009) Wang et al. (2010) Miyamoto et al. (2008) Yang et al. (2012) Hatta et al. (2002) Hatta et al. (2002) Yago et al. (2011) Segmüller et al. (2008) Segmüller et al. (2008) Segmüller et al. (2008) Schumacher et al. (2008) – Valette-Collet et al. (2003) – ten Have et al. (1998) Del Sorbo et al. (2008) Segmüller et al. (2007) – Schoonbeek et al. (2001) Zheng et al. (2000) Schoonbeek et al. (2001) Gronover et al. (2001) Dalmais et al. (2011) Dalmais et al. (2011) Frías et al. (2011) Jonkers et al. (2011) Kokkelink et al. (2011) Botrytis cinerea C. Garrido et al. Pathogen species 176 Table 10.2. List of studied genes from strawberry fungal pathogens included in the Pathogen–Host Interaction database (PHI-base) 2013. Pathogen species Phenotype of mutant PHI_ID Database Accession no. Reference BCFHG1 BcFKBP12 BcCRZ1 BcatrB bos5 BcNma BcPIE3 PGIP2 Bcchs3a bac bcpka2 bcpka1 bcpkaR bcras2 Bap1 BAC BcAtf1 Bcchs1 BCP1 BCPME1 BcPLS1 BcSOD1 BcBOT1 (CND5) BTP1 BcPG2 FRT1 BCPME2 LIP1 BCCAT2 BCATRD BCMFS1 BMP1 XYN11A CEL5A Unaffected pathogenicity Increased virulence Reduced virulence Reduced virulence Loss of pathogenicity Reduced virulence Reduced virulence Effectora Reduced virulence Reduced virulence Unaffected pathogenicity Reduced virulence Reduced virulence Reduced virulence Unaffected pathogenicity Reduced virulence Increased virulence Reduced virulence Reduced virulence Reduced virulence Loss of pathogenicity Reduced virulence Reduced virulence Reduced virulence Reduced virulence Unaffected pathogenicity Unaffected pathogenicity Unaffected pathogenicity Unaffected pathogenicity Unaffected pathogenicity Unaffected pathogenicity Loss of pathogenicity Reduced virulence Unaffected pathogenicity PHI:2304 PHI:2305 PHI:2308 PHI:2309 PHI:2319 PHI:2334 PHI:2335 PHI:2347 PHI:2359 PHI:2368 PHI:2369 PHI:2370 PHI:2371 PHI:2372 PHI:2374 PHI:240 PHI:2447 PHI:276 PHI:277 PHI:278 PHI:329 PHI:330 PHI:438 PHI:441 PHI:492 PHI:538 PHI:540 PHI:541 PHI:542 PHI:543 PHI:544 PHI:545 PHI:546 PHI:547 EMBL EMBL Uniprot Uniprot Uniprot Uniprot Genbank Uniprot Uniprot Uniprot Uniprot Uniprot Uniprot Uniprot Uniprot EMBL Broad EMBL EMBL EMBL EMBL EMBL EMBL EMBL EMBL EMBL EMBL EMBL EMBL EMBL EMBL EMBL EMBL EMBL CAP74387 ABA25866 A6RI22 Q9UW03 A6S644 E2GL14 DQ140394 Q5TIP4 Q8TG14 Q9P880 A6S414 C0H5W3 C0H5W5 A6RUJ6 D2T177 CAB77164 B0510_6765 CAA54909 AAQ16572 CAC29255 CAD43406 CAD88591 AAQ16576 CAE55153 AAV84614 AAU87358 CAD21438 AAU87359 AAK77951 CAC41639 AAF64435 AAG23132 AAZ03776 AAT40313 Turrion-Gomez et al. (2010) Melendez et al. (2009) Schumacher et al. (2008) Stefanato et al. (2009) Yan et al. (2010) Finkelshtein et al. (2011) Gioti et al. (2008) Ferrari et al. (2008) Arbelet et al. (2010) Schumacher et al. (2008) Schumacher et al. (2008) Schumacher et al. (2008) Schumacher et al. (2008) Schumacher et al. (2008) Temme et al. (2009) – Temme et al. (2012) Soulié et al. (2003) Viaud et al. (2003) Valette-Collet et al. (2003) Gourgues et al. (2004) – Siewers et al. (2005) – Kars et al. (2005) Doehlemann et al. (2005) – – – – Hayashi et al. (2002) Doehlemann et al. (2006a) Brito et al. (2006) – Continued Fungal Diseases of Strawberry Gene_name 177 Pathogen species Colletotrichum acutatum Colletotrichum gloeosporioides Gene_name Phenotype of mutant PHI_ID Database Accession no. Reference BcPIC5 BCCHS3a BOS1 BcLCC2 BCG3 TPS1 TRE1 CUTA os-1/bos1/barA BcCCC2 Ste11 Ste7 Ste50 ste12 AVR4 AOX1 Avr2 AVR2 AVR4E AVR9 ECP1 ECP2 KLAP1 Reduced virulence Reduced virulence Reduced virulence Unaffected pathogenicity Reduced virulence Unaffected pathogenicity Unaffected pathogenicity Unaffected pathogenicity Resistant to chemical Loss of pathogenicity Loss of pathogenicity Loss of pathogenicity Loss of pathogenicity Reduced virulence Effectora Reduced virulence Effectora Effectora Effectora Effector Reduced virulence Effector Loss of pathogenicity PHI:548 PHI:549 PHI:550 PHI:552 PHI:574 PHI:650 PHI:651 PHI:69 PHI:837 PHI:2483 PHI:2484 PHI:2485 PHI:2486 PHI:2487 PHI:18 PHI:199 PHI:2344 PHI:472 PHI:529 PHI:7 PHI:70 PHI:71 PHI:481 EMBL EMBL EMBL EMBL EMBL EMBL EMBL EMBL EMBL Uniprot Uniprot Uniprot Uniprot Uniprot EMBL EMBL Uniprot EMBL EMBL EMBL EMBL EMBL EMBL ABA25866 AAM14606 AAL37947 AAK77953 BAD93277 ABG25558 ABG25559 CAA93255 AAL30826 A6SEF3 B6VCT9 A6RT73 A6S3X7 B6VCT8 CAA69643 AAF82788 Q8NID8 CAD16675 AAT28197 CAA42824 CAA78400 CAA78401 AAX14039 Gioti et al. (2006) Soulié et al. (2006) Viaud et al. (2006) Schouten et al. (2002) Doehlemann et al. (2006a) Doehlemann et al. (2006b) Doehlemann et al. (2006b) – Cui et al. (2002) Saitoh et al. (2010) Schamber et al. (2010) Schamber et al. (2010) Schamber et al. (2010) Schamber et al. (2010) Westerink et al. (2004) Segers et al. (2001) van Esse et al. (2008) Rooney et al. (2005) Takken et al. (1999) Snoeijers et al. (2003) Lauge et al. (1997) Lauge et al. (1997) Chen et al. (2005) CgDN3 Loss of pathogenicity PHI:164 EMBL AAB92221 Stephenson et al. (2000) CgMEK1 (EMK1) CHIP2 CHIP3 PELB CHIP6 CAP20 Loss of pathogenicity Unaffected pathogenicity Unaffected pathogenicity Reduced virulence Reduced virulence Loss of pathogenicity PHI:165 PHI:166 PHI:167 PHI:222 PHI:243 PHI:27 EMBL EMBL EMBL EMBL EMBL EMBL AAD55385 AAD53262 AAF00024 AAD09857 AAD00894 AAA77678 Kim et al. (2000) Kim et al. (2000) Kim et al. (2000) Yacoby et al. (2001) Kim et al. (2002) Hwang et al. (1995) C. Garrido et al. Cladosporium fulvum 178 Table 10.2. Continued. Table 10.2. Continued. Pathogen species Fusarium oxysporum PHI_ID Database Accession no. Reference CgDN24 XlnR CTF1 XLNR FOXG_00076 FOXG_02277 FOXG_08661 FOXG_00016 PG1 PGX1 ARG1 FMK1 Fhk1 Fmk1 Putative zinc finger transcription factor MeaB Conserved hypothetical protein Velvet protein family MeaB AreA Msb2 Unaffected pathogenicity Unaffected pathogenicity Unaffected pathogenicity Unaffected pathogenicity Unaffected pathogenicity Unaffected pathogenicity Unaffected pathogenicity Reduced virulence Unaffected pathogenicity Unaffected pathogenicity Reduced virulence Loss of pathogenicity Reduced virulence Reduced virulence Unaffected pathogenicity PHI:521 PHI:1020 PHI:1021 PHI:1022 PHI:1101 PHI:1103 PHI:1106 PHI:1107 PHI:114 PHI:181 PHI:200 PHI:215 PHI:2252 PHI:2253 PHI:2277 EMBL Uniprot EMBL EMBL Uniprot Uniprot Uniprot Uniprot EMBL EMBL EMBL EMBL Genbank Genbank Uniprot AAB92223 A8QJI7 ABR12478 ABN41464 J9MB32 J9MHC1 J9N0G7 J9MAX2 AAC05015 AAK81847 BAB40769 AAG01162 GQ871928 AF286533 J9MB32 Stephenson et al. (2005) Calero-Nieto et al. (2007) – – López-Berges et al. (2009) López-Berges et al. (2009) López-Berges et al. (2009) López-Berges et al. (2009) – – Namiki et al. (2001) Di Pietro et al. (2001) Rispail and Di Pietro (2010) Rispail and Di Pietro (2010) López-Berges et al. (2009) Unaffected pathogenicity Unaffected pathogenicity PHI:2278 PHI:2279 Uniprot Uniprot J9MHC1 J9N0G7 López-Berges et al. (2009) López-Berges et al. (2009) Reduced virulence Increased virulence Reduced virulence Loss of pathogenicity PHI:2280 PHI:2282 PHI:2283 PHI:2284 Uniprot Uniprot Uniprot Uniprot J9MAX2 J9MHC1 J9MJU9 J9N257 Fmk1 Loss of pathogenicity PHI:2285 Genbank AF286533 ftf1 FGA1 FOW1 CHSV FGB1 foSNF1 PacC CHS2 CHS7 REN1 Unaffected pathogenicity Reduced virulence Reduced virulence Increased virulence Reduced virulence Reduced virulence Increased virulence Reduced virulence Reduced virulence Unaffected pathogenicity PHI:2307 PHI:251 PHI:254 PHI:285 PHI:300 PHI:301 PHI:315 PHI:336 PHI:337 PHI:375 Uniprot EMBL EMBL EMBL EMBL EMBL EMBL EMBL EMBL EMBL Q2V9X3 BAB69488 BAB85760 AAO49384 AAO91808 AAN32715 AAM95700 AAT77182 AAT77184 BAC55015 López-Berges et al. (2009) López-Berges et al. (2010) López-Berges et al. (2010) Pérez-Nadales and Di Pietro (2011) Pérez-Nadales and Di Pietro (2011) Ramos et al. (2007) Jain et al. (2002) Inoue et al. (2002) Ortoneda et al. (2004) Jain et al. (2003) Ospina-Giraldo et al. (2003) Caracuel et al. (2003) Martín-Udíroz et al. (2004) Martín-Udíroz et al. (2004) Ohara et al. (2004) Continued 179 Phenotype of mutant Fungal Diseases of Strawberry Gene_name 180 Table 10.2. Continued. Pathogen species Verticillium dahliae a SIX1 FGA2 FRP1 GAS1 XYL3 XYL4 FOW2 PcF Sssod1 cna1 Ss-ggt1 PAC1 VGB Ave1 VMK1 Plant avirulence determinant. Phenotype of mutant a Effector Loss of pathogenicity Loss of pathogenicity Reduced virulence Unaffected pathogenicity Unaffected pathogenicity Loss of pathogenicity Effectora Reduced virulence Reduced virulence Reduced virulence Reduced virulence Reduced virulence Effectora Loss of pathogenicity PHI_ID Database Accession no. Reference PHI:379 PHI:395 PHI:490 PHI:522 PHI:569 PHI:570 PHI:734 PHI:665 PHI:2314 PHI:2361 PHI:2411 PHI:314 PHI:2310 PHI:2331 PHI:483 EMBL EMBL EMBL EMBL EMBL EMBL EMBL EMBL Uniprot Uniprot Uniprot EMBL Genbank Uniprot EMBL CAE55866 BAD44729 AAT85969 AAX78216 AAC06239 AAK27975 BAE98264 AAK63068 A7E5X4 Q0H744 A7F946 AAF93178 JQ665433 H9DUR1 AAW71477 Rep et al. (2004) Jain et al. (2005) Duyvesteijn et al. (2005) Caracuel et al. (2005) Gómez-Gómez et al. (2002) Gómez-Gómez et al. (2002) Imazaki et al. (2007) Orsomando et al. (2001) Harel et al. (2006) Li et al. (2012) Rollins (2003) Tzima et al. (2012) de Jonge et al. (2012) Rauyaree et al. (2005) C. Garrido et al. Phytophthora cactorum Sclerotinia sclerotiorum Gene_name ©CAB International 2016 – for Amjad M.Husaini Fungal Diseases of Strawberry provide to the organization after an experiment has been validated and the role of the gene has been demonstrated sufficiently. The organization database includes not only pathogenicity factors, but also ‘loss of pathogenicity’, ‘reduced virulence’, ‘increased virulence’ or ‘unaffected pathogenicity’. A total of 25 new genes have been included in Table 10.2 since 2010. There have been 13 new B. cinerea genes. Seven genes (BcBOA2, BcBOA6, BcSpl1, BcCdc42, BcNma, Bcchs3a and Ste12) reduce virulence in B. cinerea. Four new pathogenicity factors, BcCCC2, Ste11, Ste7 and Ste50 have been described by Saitoh et al. (2010) and Schamber et al. (2010). More recently, one gene catalogued as having ‘increased virulence’ has been described by Temme et al. (2012) in this fungus. In the case of B. cinerea, the genomes of two strains have been sequenced and published, and are available in the Broad Institute database. B. cinerea has a total genome size of approximately 42 Mb, and more than 16,000 of its genes have been sequenced (Table 10.3 and Fig. 10.1). Currently, the genome is being annotated by different groups, due to the genomics information obtained from molecular studies of the fungus, including knock-out mutants, pathogenicity tests, metabolic studies and morphological characterization of mutants. There were a total of six new entries for Fusarium oxysporum in the PHI-base during 2010–2013. These included two genes that reduced the virulence, Fhk1 (Rispail and Di Pietro, 2010) and AreA (López-Berges et al., 2009); two new pathogenicity factors, Msb2 and Fmk1 (Pérez-Nadales and Di Pietro, 2011); and one gene that increases the virulence level, MeaB (López-Berges et al., 2010). This fungus is very important because of a very wide host range of crops and is included as quarantine pathogen in Europe for the EPPO agency. The sequence of the total genome is also available in the Broad Institute database, with a 61 Mb genome size and 17,708 sequenced genes, together with the sequences of two other species: Fusarium verticillioides, with a total genome size of 41 Mb and 14,169 sequenced genes; and Fusarium graminearum, 181 with a smaller genome size of 36 Mb and 13,321 sequenced genes. Another six new genes were included in the PHI-base during the 2010–2013. Yang et al. (2010) and Yago et al. (2011) described the role of the AaNoxA and AsSLT2 genes, respectively, catalogued as ‘reduced virulence’ in Alternaria alternata. This fungus can cause disease in other important crops such as pear, apple, calamondin and tobacco, among others. More than 60% of its genes, included in the PHI-base, are annotated as ‘loss of pathogenicity’ (Table 10.2). Sclerotinia sclerotiorum and V. dahliae must also be mentioned because, during 2012, three genes were included in PHIbase, and their genomes have also been completely sequenced and are available in the Broad Institute genome project database. They have a total genome size of 38 Mb (14,503 genes) for Sclerotinia sclerotiorum and 33 Mb (10,535 genes) for V. dahliae (Table 10.2 and Fig. 10.1). 10.3.6. Proteomics advances in strawberry fungal pathogens Proteomics studies have made considerable progress in recent years, accumulating relevant information about the biology of fungal pathogens. Proteomic techniques have been useful for obtaining extensive and decisive biological information about the life cycles of many organisms (Garrido et al., 2010). However, these techniques are dependent on advances in the genetic information accumulated in databases, as the result of protein sequencing must be compared with genome databases. In other cases, proteins can be identified by de novo peptide sequencing and sequence alignment (Garrido et al., 2010). Another important limitation of the techniques is the need to have an effective protein extraction method to obtain a good and representative biological sample, and the majority of proteins spots then need to be identified by two-dimensional electrophoresis for a complete proteomics study. All these limitations are progressively being overcome; we strongly recommended the specific reviews by Garrido ©CAB International 2016 – for Amjad M.Husaini 182 C. Garrido et al. Aspergillus fumigatus Aspergillus clavatus Verticillium albo-atrum Verticillium dahliae 10,221 9,887 Aspergillus fischeri 9,121 10,535 10,406 Stagonospora nodorum Aspergillus niger 12,380 11,200 Sclerotinia sclerotiorum Aspergillus oryzae 14,503 12,063 12,227 Rhizopus oryzae Aspergillus terreus 10,406 5,000 17,459 Rhizopus delemar 12,604 Aspergillus flavus 10,560 20,501 Aspergillus nidulans Phytophthora parasitica 18,179 16,448 Phytophthora infestans 12,006 Botrytis cinerea 13,321 Fusarium graminearum 17,708 14,169 16,172 Colletotrichum graminicola Colletotrichum higginsianum Fusarium oxysporum Fusarium verticillioides Fig. 10.1. Number of genes sequenced from each genome sequencing project of fungal strawberry genera available in the Broad Institute of MIT and Harvard database. Table 10.3. Genome sequence projects of fungal strawberry genera available in the Broad Institute of MIT and Harvard database. Genus Species Strain sequenced Aspergillus fumigatus clavatus fischeri niger oryzae terreus flavus nidulans cinerea graminicola higginsianum verticillioides oxysporum graminearum infestans parasitica delemar oryzae sclerotiorum nodorum dahliae albo-atrum AF293 NRRL1 NRRL 181 ATCC 1015 RIB40/ATCC 42149 NIH 264 NRRL3357 FGSC A4 B05.10/T4 M1.001 IMI349063 7600 4287 PH-1 T30-4 INRA-310 (V2) RA 99-880 1006PhL ATCC18683 SN15 VdLs.17 VaMs.102 Botrytis Colletotrichum Fusarium Phytophthora Rhizopus Sclerotinia Stagonospora Verticillium Genome size (Mb) 29.4 42.66/41.61 51.60 49.08 41.78 61.36 36.45 228.54 82.39 46.09 36.35 38.33 37.24 33.83 32.83 ©CAB International 2016 – for Amjad M.Husaini Fungal Diseases of Strawberry et al. (2010) and González-Fernández et al. (2010) for more information regarding the experimental design of proteomics studies. The biocontrol agent Trichoderma spp. is one of the microorganisms, in relation to strawberry crops, about which much biological information has accumulated using proteomics techniques. This fungus provides an abundance of biotechnologically valuable proteins and secondary metabolites. The Trichoderma harzianum and Trichoderma atroviride proteomes were first described by Grinyer et al. (2004) and subsequently by Woo et al. (2006). They concluded that several resistance genes are upregulated or activated, which allows the plant to recognize them (Woo et al., 2006). Proteomics studies have demonstrated the presence of large numbers of effective membrane pumps in Trichoderma spp., allowing their adaptation to environmental and biochemical stresses (Woo et al., 2006). Trichoderma spp. establishes a strong molecular-based communication/interaction with infected plants. The fungus induces resistance mechanisms. Three types of elicitor are produced by the fungus: (i) proteins, such as proteases, xylanases, chitinases and glucanases; (ii) Avrlike proteins; and (iii) cell wall degradation enzymes (Woo et al., 2006). In relation to phytopathogenic fungi, B. cinerea is one of the most studied fungi from the proteomics point of view. Several research groups have determined much biological information about this pathogen (Fernández-Acero et al., 2006, 2007; Shah et al., 2009a,b; Fernández and Novo, 2010). Fernández-Acero et al. (2006) reported the first proteome analysis of B. cinerea, comparing two strains with different levels of virulence (Fernández-Acero et al., 2007). Malate dehydrogenase proteins were identified and found to be overexpressed in the more virulent strain (B. cinerea CECT-2100). The role of malate dehydrogenase as a pathogenicity factor had been suggested previously, as this enzyme catalyses the reversible conversion of oxalacetate and malate; oxalacetate has been shown to be an oxalic acid precursor, which has been described as a pathogenicity factor in B. cinerea (Lyon et al., 2007). In a subsequent study, Fernández-Acero et al. (2009) analysed the proteins produced 183 by B. cinerea during cellulose degradation. A total of 267 spots were identified, and proteins were found that could play a significant role in plant infection, specifically B. cinerea peptidyl-prolyl cis-trans isomerase and glyceraldehyde 3-phosphate dehydrogenase (Fernández-Acero et al., 2009). Shah et al. (2009a) published an approach to the secretome of B. cinerea using a highthroughput liquid chromatography tandem mass spectrometry (MS/MS) approach. B. cinerea-secreted proteins were identified as transport proteins, carbohydrate metabolism proteins, peptidases and oxidation/ reduction proteins used by B. cinerea for plant infection and colonization (Shah et al., 2009a). In 2010, our group developed a proteomics approach for studying the secretome of B. cinerea using two-dimensional electrophoresis combined with MS/MS analysis. More than 70 protein spots were identified, and although many of these have not yet been functionally assigned, their regulation under the conditions assayed demonstrated their functional significance in the mechanisms of infection used by B. cinerea, which will advance our functional understanding of Botrytis pathogenesis (Fernández-Acero et al., 2010). For further details of the specific case of B. cinerea, we recommend the review by González-Fernández et al. (2010). A proteomic study of strawberry was published by Fang et al. (2012). This work tried to elucidate the defence mechanisms used by strawberry against anthracnose causal agents. The authors inoculated strawberry leaves with Colletotrichum fragariae and characterized at different times the changes in protein synthesis by the plant. They showed a dynamic overview of the altered protein expression involved in metabolism, photosynthesis, energy production, antioxidant activity and chaperone activity. The results demonstrated that there exists a protein network consisting of several functional components in the plants, including a dynamic balance between reactive oxygen species production and scavenging, accelerated biosynthesis of heat-shock proteins, impaired glycolysis and enhanced cell wall lignin formation, in response to infection by Colletotrichum fragariae. The authors ©CAB International 2016 – for Amjad M.Husaini 184 C. Garrido et al. indicated the possible existence of a systematic Colletotrichum fragariae resistance mechanism in strawberry leaves. An understanding of how plants and pathogens recognize each other and establish a successful or unsuccessful relationship is crucial for exploring the defence mechanisms of plants (Fang et al., 2012). fungal pathogens. We have described the most important diseases of strawberry crops caused by phytopathogenic fungi, and have discussed the most recent advances in the development and optimization of molecular methods and better tools for detection, identification, control and understanding of these pathogens, and their application in strawberry production fields. 10.4. Conclusions Acknowledgements More than 50 genera of fungal pathogens are known to cause diseases in Fragaria × ananassa. This crop is one of the most commercially important fruit crops in the world and is continually subjected to the risk of fungal disease, with subsequent economic losses for growers and producer countries. In recent years, many studies have focused on the development of new molecular methods, leading to a better understanding of fungal pathogens and improving the strategies available for disease control. This chapter has discussed the latest results, based on genomics, transcriptomics and proteomics approaches, for the study of strawberry We would like to thank the Spanish Government, the Ministerio de Ciencia y Tecnología, the Junta de Andalucía (Regional Government), and the EU (Fondos Europeos de Desarrollo Regional) for financing of most of the work of our group mentioned in this chapter: Spanish DGICYT AGL2006-13401-C02-02/ AGR and AGL2009-13359-C02-02; AGL201239798-C02 y AGL2015-65684-C2-2R; Junta de Andalucía, PO7-FQM-02689; and by the University of Cádiz, project UCA 18DGUE1102 V.E. González-Rodríguez was financed by grant FPU of the Ministerio de Educación, Government of Spain, ref. AP2009-1309. 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