Critical Reviews in Microbiology, 2012, 1–16, Early Online © 2012 Informa Healthcare USA, Inc. ISSN 1040-841X print/ISSN 1549-7828 online DOI: 10.3109/1040841X.2011.640977 REVIEW ARTICLE Critical Reviews in Microbiology Downloaded from informahealthcare.com by Inrs Inst Armand Frappier on 01/19/12 For personal use only. Emerging phytopathogen Macrophomina phaseolina: biology, economic importance and current diagnostic trends Surinder Kaur1,2, Gurpreet Singh Dhillon, Satinder Kaur Brar, Gary Edward Vallad3, Ramesh Chand1, and Vijay Bahadur Chauhan1 1 Department of Mycology & Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University (BHU), Varanasi, India, 2INRS, Quebec, Quebec, Canada, and 3University of Florida, Wimauma, United States. Abstract Macrophomina phaseolina (Tassi) Goid. is an important phytopathogenic fungus, infecting a large number of plant species and surviving for up to 15 years in the soil as a saprophyte. Although considerable research related to the biology and ecology of Macrophomina has been conducted, it continues to cause huge economic losses in many crops. Research is needed to improve the identification and characterization of genetic variability within their epidemiological and pathological niches. Better understanding of the variability within the pathogen population for traits that influence fitness and soil survival will certainly lead to improved management strategies for Macrophomina. In this context, the present review discusses various biological aspects and distribution of M. phaseolina throughout the world and their importance to different plant species. Accurate identification of the fungus has been aided with the use of nucleic acid–based molecular techniques. The development of PCR-based methods for identification and detection of M. phaseolina are highly sensitive and specific. Early diagnosis and accurate detection of pathogens is an essential step in plant disease management as well as quarantine. The progress in the development of various molecular tools used for the detection, identification and characterization of Macrophomina isolates were also discussed. Keywords: Epidemiology, food crops, global distribution, stem canker, taxonomy, toxin, variability Introduction the fungus to survive for prolonged periods of time in the soil (Baird et al., 2003). Besides being an opportunistic plant pathogen, several clinical reports have also established M. phaseolina as an intermittent human pathogen that may cause cutaneous and several fungal infections (Tan et al., 2008; Srinivasan et al., 2009). M. phaseolina has the potential to neutralize plant, animal, as well as human immunity in immune-suppressed patients undergoing prophylactic antifungal treatments (Arora et al., 2011). Proper identiication of M. phaseolina can often be problematic for mycologists/plant pathologists. Two asexual subphases have been documented, a saprophytic phase (Rhizoctonia bataticola) that forms microsclerotia and mycelia and a pathogenic phase (M. phaseolina) present in host tissues that forms microsclerotia, mycelia and pycnidia (Figure 1). In addition, the occurrence of Macrophomina phaseolina is a fungal pathogen with a host range of more than 500 plant families; inciting a stem canker disease in many crops that is often referred to as charcoal rot, due to the charcoal type coloration imparted to the colonized plant tissues. M. phaseolina is primarily soil borne in nature, with heterogeneous host speciicity, that is, the ability to infect monocots as well as dicots and non-uniform distribution in the soil (Mayek-Perez et al., 2001; Su et al., 2001). he pathogen is seed-borne and seed-to-seedling transmission has been documented in infected seeds (Pun et al., 1998). Macrophomina infection causes both pre- and postemergence plant mortality. Characteristic post-emergence symptoms include the development of spindle shaped lesions with dark border and light gray centre covered with small pinhead-sized microsclerotia and sometimes pycnidia. Sclerotia allow Address for Correspondence: Satinder Kaur Brar, PhD, INRS, ETE, Québec, Quebec, G1K9A9 Canada. E-mail: satinder.brar@ete.inrs.ca. (Received 17 July 2011; revised 06 November 2011; accepted 11 November 2011) 1 2 S. Kaur et al. Abbreviations Critical Reviews in Microbiology Downloaded from informahealthcare.com by Inrs Inst Armand Frappier on 01/19/12 For personal use only. AFLP, ampliied fragment length polymorphism CMI, Commonwealth Mycological Institute CWDEs, cell-wall degrading enzymes DIG, digoxigenin egl, endoglucanase gene IGS, intergenic spacers phenotypic as well as genetic variation in the pathogen population, even from the same geographical region, has been documented (Dhingra and Sinclair, 1972). his immense variation can probably be partly explained by the presence of heterokaryotic mycelium in isolates (Beas-Fernandez et al., 2006; Reyes-Franco et al., 2006). hese challenges have added to the diiculty of designing efective and sustainable disease-management strategies. In recent years, various researchers have attempted to study diferent aspects of the fungus, such as morphological, pathogenic and molecular characterization of large number of isolates with diferent geographical origin (Su et al., 2001; Purkayastha et al., 2006; Babu et al., 2007; Baird et al., 2009). Accurate identiication of the fungus has been aided with the use of nucleic acid–based molecular techniques, such as the use of species-speciic oligonucleotide primers for polymerase chain reaction (PCR) and digoxigenin (DIG) labeled DNA probes, both based on sequences of the internal transcribed spacers (ITS) (Babu et al., 2007). Experimental evidence has substantiated the role of cell wall degrading enzymes (CWDEs) (Ammon and Wyllie, 1972) and phytotoxins (Bhattacharya et al., 1992) during pathogenesis process by M. phaseolina. However, an enhanced understanding of the variability within the pathogen population for traits that inluence itness and soil survival will certainly lead to improved management ITS, internal transcribed spacers RAPD, random ampliied polymorphic DNA RFLP, restriction fragment length polymorphism ROS, reactive oxygen species SSR, simple sequence repeats VNTR, variable number tandem repeats VWC, volumetric water content. strategies for Macrophomina. he review focuses on taxonomic status, identiication of characters, symptomatology and the current status of Macrophomina diagnostics. So far, to the best of our knowledge, there is no review which describes the biology, mode of infection and epidemiology of stem canker disease caused by M. phaseolina. Taxonomy and nomenclature The taxonomic status of M. phaseolina has been revised several times over the past 100 years. The genus Macrophomina was first established by Petrak (1923) with the description of M. philippinensis from the dried specimens of Sesamum orientale collected by G. M. Reyes in Philippines in 1921. However, the pycnidial state of the fungus was originally named Macrophoma phaseolina by Tassi (1901) and Macrophoma phaseoli by Maublanc (1905). Halsted (1890) described the sclerotial state as Rhizoctonia bataticola (Taub.) Butler on sweet potato (Ipomoea batatas). Finally, Ashby (1927) critically examined and compared the type specimens of the fungus from beans with other related genera and established the binomial species Macrophomina phaseoli (Maubl.) (Ashby, 1927). Later, Goidanich (1947) changed the binomial Macrophomina phaseoli to Macrophomina phaseolina (Tassi.) Goid., since the original specimen of Macrophomina was collected by Tassi in 1901. Hence, the two names, that Figure 1. Diferent isolates of M. phaseolina on PDA: (A) aerial hyphae aggregates to form sclerotia; (B) abundant aerial hyphae; (C) most of the hyphae engrossed in the media with lesser formation of aerial hyphae; (D) abundant microsclerotia evident on the media along with the aerial hyphae and no sclerotia formation. (See colour version of this igure online at www.informahealthcare.com/mby) Critical Reviews in Microbiology Critical Reviews in Microbiology Downloaded from informahealthcare.com by Inrs Inst Armand Frappier on 01/19/12 For personal use only. Emerging phytopathogen Macrophomina phaseolina is, Macrophomina phaseoli (Maubl.) Ashby and Macrophomina phaseolina (Tassi.) Goid. became widely accepted in the literature. Additional synonyms for Macrophomina phaseolina exist in the literature including Sclerotium bataticola (Taubenh, 1913), Macrophoma cajani P. Syd. and Butler (Sydow and Butler, 1916, Farr and Rossman 2010), Macrophoma chorchori (Sawada, 1916), Macrophoma sesami (Sewada, 1922) and Tiarosporella phaseolina (Tassi; Aa, 1981). In India, the fungus was first isolated from cowpea (Shaw, 1912) and referred to as Sclerotium bataticola by Sheikh and Ghaffar (1979). During 1981, Van der Aa designated the species as Tiarosporella phaseolina (Tassi.) Van der Aa. Currently, M. phaseolina (Tassi.) Goid. 1947 is oicially recognized as the correct taxonomic name (CMI description of pathogenic fungi and bacteria no. 275) with the sclerotial phase known as Rhizoctonia bataticola (Holliday and Punithalingam, 1970). Macrophomina is a monotypic genus, composed of only one species, “phaseolina” (Sutton, 1980). Global distribution and economic importance Macrophomina phaseolina causes disease on more than 500 cultivated and wild plant species, including several economically important crops, such as legumes and vegetables. It causes stem canker, seedling blight, charcoal rot, dry root rot, wilt, leaf blight, stem blight and pre-emergence and post-emergence damping-of (Singh et al., 1990); root and stem rot of softwood forest trees (McCain and Scharpf, 1989), fruit trees and weed species (Songa and Hillocks, 1996). he fungus has a vast geographical distribution and is especially problematic in tropical and subtropical countries with arid to semiarid climates in Africa, Asia, Europe and North and South America (Gray et al., 1990; Abawi and Pastor-Corrales, 1990; Diourte et al., 1995; Wrather et al., 2001). Some of the early reports of Macrophomina disease in diferent hosts are described in Table 1. Macrophomina stem canker is ranked among the ive most important soybean diseases causing huge annual economic losses in the top-ten soybean-producing Table 1. First reports of occurrence of Macrophomina phaseolina infection in some important plants. Crop Country/continent Disease Alfalfa and white clover North America Crown rot Cactus plants (Aeonium canariense L.) Egypt Ashy stem blight (Webb & Berthelot) Canola (Brassica napus L.) Western Australia Charcoal rot Indiana and Kentucky Charcoal rot Argentina Charcoal rot Western Australia Charcoal rot Argentina Charcoal rot Coleus (Coleus forskohlii) India Root rot Coral hibiscus (H. schizopetalus) India (Kerala) Collar rot Cassava West Africa Stem rot Benin and Nigeria Preharvest root rot Cotton (Gossypium hirsutum) Georgia, USA — Guava Varanasi, India Wilt Jatropha curcas India Root rot Melon (Cucumis melo L.) Honduras Vine decline Mungbean China Charcoal rot Nattrassia mangiferae West Africa Root rot Salower Iran Charcoal rot Soda apple Florida Leaf and stem blight Soybean Iowa Charcoal rot North Dakota Charcoal rot Minnesota Charcoal rot Strawberry Northwestern Argentina Crown and root rot France Charcoal rot Spain Crown and root rot California Crown rot Israel Crown and root rot Florida Crown and root rot Sugar beet Greece Charcoal rot Sunlower Slovakia Charcoal rot Turkey Charcoal rot North and South Dakota Charcoal rot Watermelon Nagano and Kanagawa, Japan Charcoal rot © 2012 Informa Healthcare USA, Inc. 3 Reference Pratt et al., 1998 Abdel-Kader et al., 2010 Khangura and Aberra, 2009 Baird et al., 1994 Gaetan et al., 2006 Khangura and Aberra, 2009 Gaetan et al., 2006 Kamalakannan et al., 2006 Santhakumari et al., 2002 Msikita et al., 1997 Msikita et al., 1998 Baird and Brock, 1999 Dwivedi, 1990 Sharma and Kumar, 2009 Bruton, 1997 Zhang et al., 2011 Msikita et al., 1997 Razavi and Pahlavani, 2004 Iriarte et al., 2007 Yang and Navi, 2005 Bradley and Rio, 2003 ElAraby and Kurle, 2003 Baino et al., 2011 Baudry and Morzieres, 1993 Aviles et al., 2008 Koike, 2008 Zveibil and Freeman, 2005 Mertely et al., 2005 Karadimos et al., 2002 Bokor, 2007 Mahmoud and Budak, 2011 Gulya et al., 2002 Masafumi et al., 2002 Critical Reviews in Microbiology Downloaded from informahealthcare.com by Inrs Inst Armand Frappier on 01/19/12 For personal use only. 4 S. Kaur et al. countries (United States, Brazil, Argentina, China, India, Paraguay, Canada, Indonesia, Bolivia and Italy in 1998 (Wrather et al., 2001). In the USA, the annual yield suppression for crops, such as soybean was estimated to be 1.98, 0.28 and 0.49 million metric tonnes in 2003, 2004 and 2005, respectively (Wrather and Koenning, 2006). Yield losses in Phaseolus vulgaris L. in semi arid eastern Kenya was estimated up to 300 kg/ha (Wortmann and Allen, 1994). Iriarte et al. (2007) reported huge damage caused by Macrophomina in many crops in the USA. In 2009, the University of California Cooperative Extension diagnostic lab conirmed Macrophomina problems in smaller strawberry operations in Fresno and Alameda counties (Koike et al., 2009-2010). Macrophomina was irst reported to cause charcoal rot in pine (Pinus radiata D. Don) nurseries in Chile in 1983 (Butin and Peredo, 1986). M. phaseolina caused 1.7% loss of oil, 20.4% loss of dry matter and 0.1% loss of carbohydrate in groundnut (Arachis hypogaea L.) incubated on the culture plate for 7 days and 0.7% and 1.8% decrease in ash and iber contents, respectively (Umechuruba et al., 1992). In the bay region of Somalia, the incidence of M. phaseolina in sorghum ields was reported up to 70% (Gray et al., 1991). Yield losses owing to M. phaseolina infection have been estimated up to 57% in sesame (Sesamum indicum L.) at 40% disease severity (Maiti et al., 1988). M. phaseolina causes severe damage to olives in Tunisia (Boulila and Mahjoub, 1994), Egypt (Ghoneim et al., 1996), Europe and Mediterranean region (Sanchez-Hernandez et al., 1996, 1998). Identification of M. phaseolina Colony color of Macrophomina varies in culture from black to brown or gray and becomes dark in color with age. Abundant aerial mycelium is produced in the culture plate with sclerotia imbedded within the hyphae or engrossed in the agar or on the agar surface with smooth precincts. Hyphae are septate, initially hyaline turning to a honey or black color. Numerous dark brown to black colored sclerotia can be seen on the reverse side of the culture plate. he vegetative mycelium is characterized by the formation of monilid or barrel-shaped cells and the formation of septum near the branching of the mycelium. Branching occurs at right angle to parent hyphae, but branching at acute angles is also common (Dhingra and Sinclair, 1977). Microsclerotia are formed from the aggregation of hyphae with 50 to 200 individual cells coupled by a melanin pigment (Figure 2A). he microsclerotia of Macrophomina are black in color and their size varies (50-150 μm) with the host and the media used (Short and Wyllie, 1978). Pycnidia are rarely produced in the culture while it can be induced in culture by near UV irradiation for 12 h and darkness for 12 h with alternating cycle at 20-24°C. M. phaseolina generally produce globose or lattened pycnidia that range from 100 to 200 μm in diameter. Pycnidia are initially embedded in the host tissue and are dark to grayish in color but become black and erumpent with maturity (Figure 2B). he pycnidia are membranous to subcarbonaceous with a subtle or deinite truncate ostiole, solitary to gregarious and simple rod-shaped conidiophores normally 10–15-μm long, bearing single-celled conidia (Figure 2C). Ostiole is central, circular and surrounded by dark brown thickwalled cells. he pycnidia produced in the culture are typically dark brown to black, subglobose to lageniform with the diameter of around 300 μm and composed of several layers of cells. he inner layer is hyaline while the outermost layer Figure 2. (A) Germinating microsclerotia isolated from pigeonpea and (B & C) pycnidiospores protruding out from pycnidia on pigeonpea stem. (See colour version of this igure online at www.informahealthcare.com/mby) Critical Reviews in Microbiology Emerging phytopathogen Macrophomina phaseolina of cells is dark brown to black in color. Conidiophores are septate or branched and simple. Conidia are characteristically hyaline, obovoid, with truncate base that ultimately becomes rounded and measures around 5–10 × 14–30 μm (Punithalingam, 1982). he apex is usually rounded and covered with a thin membrane that evert and stay at the apex and measures up to 16–24 × 5–9 μm. Eversion and gelatinization of the outer layer of conidial wall results in the formation of apical and cap or funnel-shaped conidial appendage. Critical Reviews in Microbiology Downloaded from informahealthcare.com by Inrs Inst Armand Frappier on 01/19/12 For personal use only. Symptomatology Spindle-shaped lesions with light gray centers and brown margins with scattered pycnidial bodies are the early symptoms of Macrophomina infection in woody plants. Generally Macrophomina can cause a range of symptoms after a successful infection from restricted spindle-shaped lesions on the stem to extended lesions that result in the wilting of the plant. Deep and irregular necrotic lesions extending toward hypocotyls and root surfaces were observed in soybean (Ammon and Wyllie, 1972), chickpea (Singh and Mehrotra, 1982) and sorghum (Pedgaonkar and Mayee, 1990). As lesions coalesce they form larger patches on branches or entire plant, leading to premature senescence and plant death (Dhingra and Sinclair, 1978). During the early stages of disease development, several black dots like pycnidia are evident on these lesions. Later on, several pycnidia can be observed on most of the infected parts of the plant. Infected plants dry up and the roots are decayed with a shredded appearance (Ilyas and Sinclair, 1974). Infected ine roots develop dark sclerotial bodies and characteristic dark, blackened streaks can be seen when peeled of. Symptoms on Phaseolus vulgaris L. are characterized as small lesion in the epidermal and external cortex cells of hypocotyls. Microsclerotia germinate and produce infection hyphae which then penetrate through the epidermal cells and grow intercellularly afterward. he infection results in the cellular collapse, necrosis of epidermal and cortex cells in roots and hypocotyls in common bean (Mayek-Perez et al., 2002). Colonization of the epidermis and the cortical cells is followed by the colonization of the vascular cambium and phloem cells in chickpea (Singh et al., 1990), garlic (Dwivedi et al., 1994), maize (Singh and Kaiser, 1994) and guava (Suarez et al., 1998). Typical symptoms incited by M. phaseolina with reference to some crops are described in Table 2. Usually, the symptoms develop later in the season as plants begin to lower; however, Macrophomina can even Table 2. List of diseases caused by Macrophomina phaseolina. Disease Host Symptoms Wilt or dry Cicer arietinhum Disease most prevalent in hot dry climate during post-lowering, root rot favorable temperature 30°C; tap root dries up and devoid of ine roots; numerous dark pinhead sclerotia develops underneath bark. Cajanus cajan Disease becomes more aggressive during summer; stunting of plants; tap root becomes brittle; small gray black sclerotia develops in the root pith. Phaseolus vulgaris Symptoms appears at the seedling stage; pinhead spots develops that Damping coalesce later on and become water soaked; brown to black thin streaks of and Vigna mungo develops on the collar region that results in the seedling collapse. Eucalyptus Seedlings are infected, leaves turns yellowish brown to black; necrotic Seedling blight lesions develops on the upper part of the stem; stem breaks at the collar region. Sorghum Plants get infected at growth stages of the crop, colonization irst Charcoal rot occur through the roots; xylem vessels are blocked; and the tissues disintegrate. Glycine max 30–38°C is the most predisposable factor for disease development; 32–77% disease incidence reported; black spots and blemishes develop (Soybean) on the infected seed coat; seed emergence is reduced. Melon Crown leaves turn yellow and withers; water-soaked lesion develops near the ground; amber colored gum is produced. Vigna unguiculata Typical symptoms appear at unifoliate leaf stage; pinhead-size, charcoal-colored spots appear; mostly restricted to the seed (L.) Walp. hypocotyls. Infected spots expand and develop into large necrotic (Cowpea) lesions resulting in death of the plant; early infested grains abort and dry; pods shrunken, deformed and thin. Root rot Phaseolus vulgaris Root system becomes rusty brown it appears at early stages of the crop development and is recognized by dropping and wilting. and Vigna mungo Gossypium spp. Disease appears at early development stages; symptoms evident after lowering; causes root rotting; development of necrotic lesions on the afected parts; several ine sclerotia seen on diseased afected tissue; sudden withering of plants. Crown rot Strawberry Dark brown necrotic areas develop on the margins of the cut crowns of and root rot diseased plants along the woody vascular ring; roots become necrotic. © 2012 Informa Healthcare USA, Inc. 5 References Leach and Garber, 1970 Noble and Richardson, 1968 Chandra et al. 1995 Soni et al., 1985 Parmeshwarappa et al., 1976; Seetharama et al., 1987; Chandra et al., 1995 Gangopadhyay, 1973 Etebarian, 2006 Bouhot, 1967; Adam, 1986 Jhooty and Brains, 1972 Novotelnova, 2005 Aviles et al., 2008 6 S. Kaur et al. infect the roots of emerging seedlings leading disease to seedling blight (Machado, 1987). Infected seedlings show reddish brown discoloration on the soil line extending up the stem that may turn dark brown to black. Pycnidia are rarely produced on the host whereas mycelia and sclerotia are abundantly formed (Knox-Davies, 1966). he development of a gray or silver color can be seen on pods, petioles, stems, and roots in soybean that becomes evident owing to the formation of microsclerotia in the infected tissues (Wyllie, 1988). Critical Reviews in Microbiology Downloaded from informahealthcare.com by Inrs Inst Armand Frappier on 01/19/12 For personal use only. Epidemiology Epidemiology is the simultaneous study of pathogen population with the host populations in their natural environment commonly termed as disease triangle. he presence of a susceptible host, virulent pathogen and environmental conditions conducive for the disease development represents a perfect disease triangle. he disease cycle for M. phaseolina is presented in Figure 3. Microsclerotia produced in the roots and stem tissues of its hosts serve as the primary source of inocula and can persist in the soil for around 15 years (Short et al., 1980). Microsclerotia have been reported up to the depth of 0–20 cm in soil but are generally found in clusters on the soil surface (Alabouvette, 1990; Campbell and Van der Gaag, 1993) and are well adapted to survive under adverse environmental conditions, such as low soil nutrient levels and temperature above 30°C which prevail in tropical and subtropical countries (Short et al., 1980). he germination of microsclerotia occurs frequently in the temperature range of 28–35°C (Mihail, 1989). An appresoria formed from the germ tubes penetrates through the host epidermis by secreting CWDEs while indirect penetration through natural openings or wounds has also been reported (Bowers et al., 1999; Mayek-Perez et al., 2002). he ramifying mycelia colonize the vascular tissue by growing through the cortex and then entering the xylem vessels (Abawi and Pastor-Corrales, 1990). Once inside vascular tissues, the fungus spreads through the tap root and plugs the vessels resulting in wilting of the plant (Wyllie, 1988). Toxin production and enzymatic degradation may also lead to wilting (Jones and Wang, 1997; Kuti et al., 1997). Under natural conditions, pycnidia are rarely observed on their respective hosts but can be induced in vitro by providing diferent incubation conditions (Mihail and Taylor, 1995; Gaetan et al., 2006). he ability of Macrophomina to produce pycnidia depends on the host and the speciic nature of the fungal isolate, which will determine the epidemiological role of conidia in the disease cycle (Ahmed and Ahmed, 1969). Targeting the microsclerotia in the soil or pycnidia may help in reducing the primary inoculum and the rate of secondary spread, respectively, and hence slow disease progress. he survival of Macrophomina was reported from 2–15 years and was largely inluenced by environmental conditions irrespective of association with the host tissues (Short, 1980; Baird et al., 2003). he fungus survived in cucurbit roots, sorghum and corn Figure 3. Diagrammatic representation of the disease cycle for the stem canker caused by Macrophomina phaseolina. (See colour version of this igure online at www.informahealthcare.com/mby) Critical Reviews in Microbiology Critical Reviews in Microbiology Downloaded from informahealthcare.com by Inrs Inst Armand Frappier on 01/19/12 For personal use only. Emerging phytopathogen Macrophomina phaseolina residues under dry soil conditions for up to 10, 16 and 18 months, respectively (Ghafar and Akhtar, 1968; Cook et al., 1973). Repeated freezing and thawing of soil, low carbon to nitrogen ratios in soil, and soil moisture content are the most important factors that signiicantly afect microsclerotia survival (Dhingra and Sinclair, 1975). Dhingra and Sinclair (1977) and Olaya and Abawi (1996) documented the enhanced production of microsclerotia under low water potentials that occurs during drought. High soil moisture has proved detrimental and reduced the survival of M. phaseolina sclerotia in soil (Dhingra and Sinclair, 1975). Based on the half-life concept (Eq. 1), Masafumi et al. (2002) forecasted that the population density of the fungus in Nagano soil after 6 years of storage was 1 per gram of soil and t1/2 was 1.88 and P0 was 9.1 (Masafumi et al., 2002). t1/2 = T × log 2 / log P0 − log P1 (1) where P0 = original population; P1 = population after time T. According to this concept, the longevity of the pathogen was estimated from the original population at certain time intervals. It is deined as the time interval for half of the individuals to be exhausted or half-life of the population. In order to better manage Macrophomina, it is important to understand sclerotia biogenesis and the factors that determine its survival in the soil. he present status of information suggests that several factors are involved in sclerotium biogenesis, such as nutritional, nonnutritional, speciic metabolite(s) and morphogenetic factors (Georgiou et al., 2006). his implicates the fact that the growth factors involved in elimination or promotion of oxidative stress are anticipated to inhibit or promote sclerotium biogenesis, respectively. For example, antioxidant growth factors, such as vitamins (ascorbic acid), microelements (selenium) and sulfhydryl compounds, such as L-cysteine, L-cystine, L-homocysteine, glutathione, thioglycolic acid and 2-mercaptoethanol do not encourage sclerotia formation in Sclerotium rolfsii (Hadar et al., 1983; Georgiou et al., 2003). hese are wellknown free-radical scavengers which minimize oxidative stress. Lipid content varies considerably in mycelia and microsclerotia produced by M. phaseolina. Lipids are known to sustain low rates of respiration under adverse conditions for long period of time as well as provide energy for germination of the dormant and resistant propagative units, such as sclerotia (Wassef et al., 1974). Wassef et al. (1985) studied the polar lipids of Macrophomina and reported that phosphatidic acid and phosphatidyl glycerol were major components of sclerotia, whereas phosphatidyl ethanolamine and phosphatidyl inositol were the major phosphatides of mycelia. Storage lipids are neutral and serve as carbon and energy reserves. Sclerotial bodies are rich in neutral lipids, and hence they are able to survive as dormant structures (Wassef et al., 1974). Complete © 2012 Informa Healthcare USA, Inc. 7 lipid proiling may enable understanding the molecular basis of the survival mechanisms of resting propagules, such as sclerotia. he outcome of these studies opens new avenues for developing non-toxic antioxidant control protocols. he use of phospholipases for disease management provides an alternative to toxic fungicides. Seeds and crop debris play an important role in the growth, development and survival of the pathogen and must be carefully studied (Baird et al., 1993; Sumner et al., 1995). Seeds, diseased plant tissues and root stocks may harbor the pathogen and may be disinfected prior to sowing by surface sterilization. However, it must be ensured that the pathogen has not become systemic. he use of disease-free seeds is also likely to reduce the level of initial inoculum and hence decreased disease severity. he nature of survival of Macrophomina also explains the spatial distribution of the pathogen over a wide geographical area. herefore, it suggests the importance of cultural exclusion to prevent the introduction of the pathogen and the systematic inspection of traded plant materials to minimize the spread of the pathogen. Cell-wall degrading enzymes (CWDEs) in M. phaseolina Macrophomina has the unique property to colonize living as well as the dead tissues (Wang and Jones, 1995). he invasion and penetration of host cell by M. phaseolina is facilitated by an array of CWDEs. Endoglucanases have been reported as one of the most important enzymes involved in pathogenesis caused by M. phaseolina (Heiler et al., 1993). Wang and Jones (1995) reported the presence of unique β-1, 4-endoglucanase similar to those endoglucanases found in plants, which may explain how the fungus can penetrate the plant cell wall so efectively. Of the two endoglucanase encoding genes, egl1 and egl2, characterized from Macrophomina, egl2 possessed amino acid and enzymatic similarity with egl3 from Trichoderma reesei (Saloheimo et al., 1988; Wang and Jones, 1995). he endoglucanase egl1 difered from other characterized endoglucanases found in saprophytic fungi, based on nucleotide sequence (Wang and Jones, 1995), substrate speciicity (Hatield and Nevins, 1987) and the lack of a cellulose binding domain and a linker (Gilkes et al., 1991). Although the cellulose binding domain is not required for pathogenesis, it increases substrate speciicity to incorporate microcrystalline cellulose. EGL1 requires four adjacent β-1, 4 linkages whereas all known endoglucanases associated with saprophytic fungi require only three contiguous linkages. Interestingly, this substrate requirement has previously been found for an auxin-responsive plant endoglucanase which is active during cell-wall expansion (Hatield and Nevins, 1987). It is possible that EGL1 attacks mixed linkage β-glucans or xyloglucans in a manner similar to plant endoglucanases leading to cell-wall loosening rather than more extensive hydrolysis, as is common for saprophyte-speciic endoglucanases (Carpita and Gibeaut, 1993). Critical Reviews in Microbiology Downloaded from informahealthcare.com by Inrs Inst Armand Frappier on 01/19/12 For personal use only. 8 S. Kaur et al. Several other hydrolytic enzymes, such as amylases, hemicellulases, pectinases, proteases and phosphatidases have also been reported to play a crucial role in disease development (Amadioha, 2000). Proteins and phosphatide lipids are the primary components of the biological membranes. he enzymes degrading such molecules may play an important role in pathogenesis, such as phosphatide degrading enzymes. hese enzymes target the cellular organelles, such as mitochondria, and alter their permeability. Many lysosomal enzymes, such as pectinases, are also released as a result of compartmentalization of such organelles. Several plant pathogenic fungi extensively utilize a cocktail of CWDEs including phosphatidases. he role of phosphatidases in the manifestation and progression of disease was studied in diferent cultivars of Brassica juncea plants challenged with M. phaseolina (Srivastava and Dhawan, 1982). An increase in the phosphatidase activity of Macrophomina was only observed on susceptible cultivars, suggesting that the expression level of these pathogenicity factors (enzymatic genes) are related to host susceptibility and could be used to not only asses host resistance but perhaps virulence (Srivastava and Dhawan, 1982). More recently, the ability of carbohydrate degrading enzyme production by two diferent isolates of M. phaseolina (microsclerotial (MphP) and mycelial (MphM)) was studied on apple pomace supplemented with 1% (w/w) rice husk as a substrate through koji fermentation (Kaur et al., 2011). Among the two isolates, MphP was observed as a potential source of diferent hydrolytic enzymes, such as cellulases, hemicellulase and amylase as compared with MphM. his study reported for the irst time the potential of hydrolytic enzyme bioproduction by diferent isolates of M. phaseolina. Higher enzyme production studies by Macrophomina may lead in new directions with respect to modulating efective plant protection strategies as well as higher production of industrially important enzymes. his study may serve to increase the understanding of diferent substrates that efect the production and role of fungal cellulases in phytopathogenicity. Toxin production by Macrophomina he phytotoxin was irst identiied from the culture iltrates of M. phaseolina (Siddiqui et al., 1979). Later, Dhar et al. (1982) described the detailed structure of the phytotoxin which is an eremophilane sesquiterpenoid, speciically an epoxidized analogue of phomenone. Kitahara et al. (1991) synthesized it semisynthetically from (+)-sporogen-AO. he toxin afects the germination of seed (50%) even at a low concentration of 0.60–2.1 pg/g wet tissue (Bhattacharya et al., 1992, 1994). he percentage seed germination has been correlated with the amount of toxin (Dhar et al., 1982; Bhattacharya et al., 1987). Enzyme-linked immunosorbent assay (ELISA) was used for the isolation and identiication of the exotoxin secreted by M. phaseolina in the diseased plant tissues (Bhattacharya et al., 1992). Several phythopathogenic fungi secrete one or more phytotoxins that facilitate host penetration, invasion and colonization. his is one of the commonly used strategies to incite disease in plants. Mycotoxin biosynthetic genes regulating mycotoxin biosynthesis seems to be acquired as units similar to antibiotic biosynthetic gene packages in Streptomycetes (Shier et al., 2007). Several phytotoxic metabolites produced by M. phaseolina have been identiied and related to the virulence of individual isolates (Bhattacharya et al., 1992). Mycotoxins produced by M. phaseolina include asperlin, isoasperlin, phomalactone, phaseolinic acid, phomenon and phaseolinone (Dhar et al., 1982; Mahato et al., 1987). Phaseolinone is a non-host-speciic heat-resistant exotoxin and inhibits seed germination in black gram (Phaseolus mungo) at a concentration of 25 µg/ml (Bhattacharya, 1987). It also causes symptoms of wilting in seedlings and necrotic lesions on leaves in a manner similar to those incited by the pathogen in a number of hosts (Bilgrami et al., 1979). However, the possible role of toxin(s) in disease development is not clearly explained. An array of toxins are produced by a large number of fungal plant pathogens but their detailed mechanism of synthesis, production and interaction with the host cellular machinery is scanty (Ballio, 1991). Mycotoxins may play an important role either in the suppression of the induced resistance or in the activation of the plant response (Berestetskiy, 2008). UV-mutated non-toxigenic, avirulent mutants of M. phaseolina and a human and animal pathogen, A. fumigatus were reported to cause infection in Phaseolus mungo seedlings only in the presence of phaseolinone (Sett et al., 2000). his study conirmed phaseolinone as a major phytotoxic substance produced by M. phaseolina in disease initiation. Phaseolinone seemed to reduce the immunity in the seeds in a non-speciic manner. However, the role of phaseolinone in the initiation of root infections by M. phaseolina remains unknown. Conlicting reports occur in literature regarding the nature and type of mycotoxin produced by M. phaseolina associated with charcoal rot disease isolated from India (Asia) in 1979 and in Mississippi (North America) in 2007. Ramezani et al. (2007) reported another phytotoxin named botryodiplodin from M. phaseolina in Mississippi; however, no phaseolinone was detected from the fungus. Botryodiplodin has been reported from the culture iltrates of several other fungi, such as Penicillium stipitatum (Fuska et al., 1974), Apiosordana sp., Lancunspora tetraspora, Triangularia bambusae, Zopiella matsushimae (Nakagawa et al., 1979), P. carneolutesens (Fujimoto et al., 1980) and P. roqueforti (Moreau et al., 1982). Botryodiplodin was irst isolated from culture iltrates of a cellulolytic fungus from mildewed tent fabric in India (isolated in 1944) by Sen Gupta and colleagues (1966). he fungus was identiied by the CMI as Botryodiplodia theobromae Pat. (syn. Lasiodiplodia Critical Reviews in Microbiology Critical Reviews in Microbiology Downloaded from informahealthcare.com by Inrs Inst Armand Frappier on 01/19/12 For personal use only. Emerging phytopathogen Macrophomina phaseolina theobromae [Pat.] Grifon & Maubl). he fungus is an important phytopathogen around the globe and infects several economically important crops in the tropics and subtropics. he possible justiication for the obvious inconsistency regarding the type of mycotoxin produced by M. phaseolina as suggested by Shier et al. (2007) is as follows: (i) Divergent evolution on the two continents, Asia and North America, around 255 million years ago, that resulted in the acquisition of diferent gene packages regulating mycotoxin biosynthesis. Moreover, the properties of phaseolinone described by Siddiqui et al. (1979) actually designates (–)-botryodiplodin; (ii) M. phaseolina might have produced two mycotoxins, (–)-botryodiplodin and phaseolinone, where partial structural characterization on one (botryodiplodin) was done by Siddiqui et al. (1979) while structure elucidation was conducted on other phytotoxin (phaseolinone) by Dhar et al. (1982). Some biochemical similarities occur in both the toxins, (–)-botryodiplodin and phaseolinone, such as both posses keto as a functional group that can give colored derivatives with 2,4-dinitrophenylhydrazone. Both the toxins produce colored reaction products with 4-(p-nitrobenzyl) pyridine, an epoxide chromogenic reagent although (–)- botryodiplodin must react by a Schif’s base, rather than an electrophiles (Penketh et al., 1994). Most of the researchers have reported phaseolinone as a mycotoxin produced by M. phaseolina. However, the occurrence of contradictory reports mandates further research to elucidate M. phaseolina toxin and its analogues. Variability in M. phaseolina Fungi with a sexual phase can generate variation through genetic recombination, whereas those fungi, such as M. phaseolina, without a known sexual phase must rely on mutations, hyphal fusion and mitotic recombination to generate genetic variation (Carlile, 1986). he success of modern agriculture relies on the ability to produce a uniform, homogeneous crop across a large geographic area. Although eicient, the modern agricultural system also puts enormous selection pressure on the pathogen to adapt to the existing environment; including genetic resistance in the crop or fungicides deployed to control the pathogen. herefore, it is important to understand the level of genetic variation that exists in fungal populations and the rate at which this variation can be generated to understand how fungi adapt to their environment. his knowledge is important to improve the development and deployment of resistant crop varieties and to modulate disease-control methods. In spite of being a mono-speciic genus, Macrophomina exhibits a high degree of morphological (Mayek-Perez et al., 1997), pathogenic (Mayek-Perez et al., 2001; Su et al., 2001), physiological (Mihail and Taylor, 1995) and genetic (Vandemark et al., 2000; Mayek-Perez et al., 2001; Pecina-Quintero et al., 2001; Su et al., 2001; Almeida et al., 2003; Jana et al., 2003, 2005a, 2005b) variation. © 2012 Informa Healthcare USA, Inc. 9 Due to high intraspeciic morphological and pathogenic variability, M. phaseolina could not be characterized into special forms, subspecies or physiological races (Echavez-Badel and Perdomo, 1991; Purkayastha et al., 2003). Several studies have been carried out to understand the variability on the basis of geographical origin (Reyes-Franco et al., 2006). Chlorate, an analogue of nitrate, is reduced to chlorite via the nitrate reductase pathway and can be toxic to fungi and plants (Williams and Miller, 2001). Characterization of the isolates on the basis of chlorate utilization was reported by several authors (Su et al., 2001; Purkayastha et al., 2006). According to Pateman and Kinghorn (1976), unrestricted growth of the isolates was the result of inactivation of one or more of the ive enzymes involved in nitrate reductase pathway. hese isolates are termed as nitrogen mutants and have been used in vegetative compatibility studies in various plant pathogenic fungi. Inorganic nitrate is utilized as a sole nitrogen source by M. phaseolina and many other fungi unless alternative nitrogen sources, such as ammonia, glutamine or glutamate are deicient (Dhingra and Sinclair, 1978;Marzluf, 1997). Later on Pearson et al. (1986) successfully utilized potassium chlorate as a phenotypic marker for identifying host-speciic isolates of M. phaseolina. he authors proposed that unlike chlorate resistant isolates, chlorate sensitive isolates were unable to utilize the same nitrogenous compound. he isolates produced only sensitive (restricted or feathery) or resistant (dense) phenotypes, when grown on minimal medium amended with 120 mM potassium chlorate. he chlorate-resistant corn isolates were distinguished from chlorate-sensitive soybean isolates obtained from soybean roots or soybean ield soils which were later conirmed by other researchers (Su et al., 2001; Purkayastha, 2006). he chlorate sensitive isolates eiciently reduced potassium nitrate to nitrite whereas resistant isolates failed to utilize it as a sole source of nitrogen. his suggests that chlorate resistant isolates lack the nitrate reductase pathway (Su et al., 2001; Purkayastha, 2006). Nitrate reductase may act as an allosteric enzyme, in the presence of certain amino acids, in association with their respective host (Garrett and Amy, 1978). Since chlorate-sensitive isolates utilize nitrate along with other nitrogen sources, higher disease severity was reported in sorghum (Das et al., 2008). However, the available reports suggest that there is no correlation between the chlorate sensitivity and host speciicity as well as with ield virulence. herefore, the chlorate phenotype might not be useful for studying host specialization studies in M. phaseolina. Further studies on genetic basis for chlorate sensitivity can generate more information on the role of nitrate utilization in studies related to pathogenicity. hese studies can contribute in understanding the pathogenesis as well as pathogenic variability. Pathogenic variability has been reported from Macrophomina isolates originating from soybean, sunlower, groundnut and common bean (Dhingra and Sinclair, 1972; Sobti and Sharma, 1992; Mayek-Perez Critical Reviews in Microbiology Downloaded from informahealthcare.com by Inrs Inst Armand Frappier on 01/19/12 For personal use only. 10 S. Kaur et al. et al., 2001). Diferent isolates of M. phaseolina from various ields cultivated with soybean, cotton, sorghum and maize exhibited variations in pathogenicity based on the cropping history and suggest that the fungus has some level of host specialization (Su et al., 2001). Similarly, Almeida et al. (2003) analyzed isolates from soybean, sorghum, sunlower, cowpea, corn and wheat as well as soil samples from native areas and reported that crop cultivation following crop rotation tends to reduce the level of host specialization within M. phaseolina ield populations. Similar to the classiication of races of anthracnose, Mayek-Perez et al. (2001) proposed a set of bean diferentials (12 cultivars) while assigning a binary value to each cultivar in order to establish a method for the characterization of pathogenicity patterns of M. phaseolina. Jana et al. (2003,2005b) reported novel and precise strategies for diferentiation of M. phaseolina isolates on the basis of host or geographical origin using random ampliied polymorphic DNA (RAPD), simple sequence repeats (SSRs) or universal rice primer PCR (URP-PCR). High degree of polymorphism was observed among the isolates of M. phaseolina isolated from the same geographical location which conirms the existence of high genetic variability (Rajkumar and Kuruvinashetti, 2007). Close genetic relationship was found among the isolates even when isolated from the same locations (Das et al., 2008). RAPD analysis of the Macrophomina isolates from the speciic host showed similar patterns but diferentiated from the isolates of other hosts. RAPD markers have proved to be an excellent tool in measuring genetic relationship and variation within and among populations of M. phaseolina. Various researchers have demonstrated the eicient utilization of RAPD analysis to study and correlate the ecology and biology of the fungus (Jana et al., 2003; Purkayastha, 2006). Purkayastha et al. (2006) investigated the applicability of RAPD and RFLP of ITS region to analyze the genetic variation among the isolates of M. phaseolina from cluster bean as well as other host plants. hey reported that the RAPD patterns can distinguish the isolates on the basis of chlorate phenotype and the host origin. Studies conducted by Su et al. (2001) and Almeida et al. (2003) indicated that restriction analysis of the ITS region might be unsuitable for detecting variability in M. phaseolina isolates from corn, cotton, sorghum and soybean. he molecular polymorphism obtained was largely independent of geographical origin. However, studies conducted by Su et al. (2001) and Babu et al. (2007) revealed high homogeneity in ITS conserving sequences among the M. phaseolina isolates irrespective of their host speciicity. Reyes-Franco et al. (2006) successfully diferentiated the isolates of M. phaseolina representing diferent geographical locations using ampliied fragment length polymorphism (AFLP). Arbitrary Simple Sequence Repeat (SSR) motifs and sequence-based SSR loci have been established as an excellent tool for the evaluation of isolates of diferent geographical origin and host variability (Jana et al., 2005a; Purkayastha et al., 2008; Baird et al., 2009). SSR loci can be transferred from one genus to another closely related genus and can be used to study the gene low within family, such as the genera Botryosphaeria and Diplodia which are closely related to the genus Macrophomina (Crous et al., 2006). he genome of M. phaseolina comprises microsatellite core sequence repeats, such as (CAC) 5, (ACTG) 4 and (GACAC). Within the genome, these are frequently interspersed repeats and can be used as an important marker in epidemiological, genetic and population studies (Jana et al., 2005a). Moderate to high genetic diversity between the isolates collected from soybean was pointed by Baird et al. (2009) and the development of 46 primer pairs (SSR loci) out of which 12 were most polymorphic. However, SSRs have disadvantage of sometimes amplifying homologous loci. Peakall et al. (1998) reported 50–100% ampliication of SSR loci between species within genera in plants whereas it was 34% within genera in fungi as described by Dutech et al. (2007). Alvaro et al. (2003) and Jana et al. (2003) observed genetic similarities among the isolates of diferent geographical locations. Such relationship among the indigenous population supports the origin from the common ancestors that has infected various crops over generations. It occurred either due to the germplasm exchange, import of infected seeds or equipment as well as by the soil infected with sclerotia. Current status in M. phaseolina diagnostics Species-speciic primers and probes have been used for the identiication and detection of M. phaseolina at molecular level by exploiting rDNA gene cluster as a target. he primers, MpKF1 (5′-CCG CCA GAG GAC TAT CAA AC-3′) and MpKR1 (5′-CGT CCG AAG CGA GGT GTA TT-3′) designed from the conserved sequences of the ITS region was highly speciic and yielded a speciic 350 bp products. Since, this 350 bp amplicon was absent from other soil-borne pathogens, it can be used for the species-speciic identiication for M. phaseolina (Babu et al., 2007) (Figure 4). An oligonucleotide probe, MpKH1, have been designed at the ITS region, and it can identify the target sequences even at minute concentration (Babu et al., 2007). hese probes are able to detect the speciic sequences even from the soil DNA. However, since these probes have shown false positive results, due to the presence of 20 bp, it might be used with caution. he quantiication of M. phaseolina from soil and plants by real-time PCR by using Taq Man and SYBR Green assay techniques are now available. Realtime quantitative PCR primers for the detection and quantiication M. phaseolina from rhizospheric and diseased plant tissues were developed by Babu et al. (2011). MpSyK and MpTqK speciic primers were designed for SYBR green and TaqMan assays, respectively, by targeting ~1 kb sequence characterized ampliied region (SCAR) of M. phaseolina. he least detection limit of TaqMan assay was up to 30 fg/µl of M. phaseolina DNA Critical Reviews in Microbiology Emerging phytopathogen Macrophomina phaseolina 11 Critical Reviews in Microbiology Downloaded from informahealthcare.com by Inrs Inst Armand Frappier on 01/19/12 For personal use only. and quantiication limit of M. phaseolina viable population was approximately 0.66 × 105 CFUs/g soil equivalent to 10 pg/µl of target DNA. Increasing sequence data in public domains has facilitated the detection systems of plant pathogens such as M. phaseolina due to incorporation of large number of isolates. Table 3 shows the recent database showing the advancement/milestones in Macrophomina research. In depth studies are required to make isolate-speciic molecular markers. hese markers can aid in epidemiological studies leading to the understanding of disease control strategies. Such identiication or detection kits, Figure 4. Development of speciic oligonucleotide primers and probe for M. phaseolina: Alignment of ITS-1 and ITS-1 sequences from eight isolates (DQ359737-DQ359744) of M. phaseolina and two reference sequences (AF132795 and U97333) taken from Gene Bank database. Color bars designate the diferent nucleotides (A-red, G-yellow, T-blue and C-green). he regions 1, 2, 3 and 5 are completely aligned, 5.8 S RNA gene is not shown and because of variability the region 4 was omitted. he position of the irst nucleotide of region 1 and others were given according to the reference sequence AF132795 (Babu et al., 2007). (See colour version of this igure online at www.informahealthcare.com/mby) developed from variety of DNA sequences including RAPD-SCAR or speciic genes coding for rRNA, will improve the detection methods for plant protection as well as quarantine authorities. Conclusion and future perspectives Increasing incidence of plant diseases create challenging problems and pose real economic threat to agricultural ecosystems. Despite the widespread use of chemicals and fertilizers, the losses due to disease continue to be signiicant. Characterization of the pathogen on various aspects, such as morphology and genetic makeup, will provide ample opportunities to understand the population genetics and itness parameters. Characterization on the basis of morphology and cultural characteristics yielded insigniicant variations between pathogenic and non-pathogenic isolates of the fungus. herefore, a rapid, sensitive and cost-efective method is required for the identiication, characterization, screening and monitoring of the pathogenic or nonpathogenic population. More studies are needed to determine the temporal and spatial distribution of the pathogen. Proper characterization of symptoms of the disease for timely scouting is needed. hese additional studies could assist the growers in understanding and managing emerging stem canker disease. Evaluation of the genetic diversity of M. phaseolina isolates from some parts of the world has been an initial step toward understanding the population structure of M. phaseolina. he RAPD-PCR technique is an important tool in discerning genetic diversity within and among groups of isolates from different hosts. Table 3. Database of the progress in M. phaseolina research (Source: NCBI Database) Source Information Books Online books Nucleotide Core subset of nucleotide sequence records EST Expressed sequence tag records GSS Genome survey sequence records Protein Sequence database Genome Whole genome sequences Structure hree-dimensional macromolecular structures Taxonomy Organisms in GenBank SNP Single nucleotide polymorphism dbVar Genomic structural variation Gene Gene-centered information SRA Sequence read archive BioSystems Pathways and systems of interacting molecules HomoloGene Eukaryotic homology groups Probe Sequence-speciic reagents UniSTS Markers and mapping data PopSet Population study data sets GEO DataSets Experimental sets of GEO data PubChem BioAssay Bioactivity screens of chemical substances PubChem Compound Unique small molecule chemical structures PubChem Substance Deposited chemical substance records Protein Clusters Collection of related protein sequences © 2012 Informa Healthcare USA, Inc. Remarks — 917 176 46 11 None None 1 None None None None None None None None 22 None 11 None None None Critical Reviews in Microbiology Downloaded from informahealthcare.com by Inrs Inst Armand Frappier on 01/19/12 For personal use only. 12 S. Kaur et al. Better understanding of M. phaseolina population diversity will assist breeders in optimization of breeding strategy that will enable long-term resistance over broader geographical areas. Till date, gene expression analysis and pathogenicity genes in Macrophomina have not been identiied, characterized and sequenced. Apparent understanding of the enzymes, particularly hydrolytic enzymes can provide better picture of the infection process or pathogenic ability possessed by M. phaseolina. he pathogenic and genetic variability has been described earlier; however, still there are some lacunae to be illed for instance, the evolutionary background, adaptability to wide host range, isolation, identiication and characterization of pathogenicity genes. he identiication of traits depicting disease resistance to stem canker would enhance the resistance breeding program. he production of toxin by diferent isolates of the fungus in natural conditions should be studied in detail to get the clear understanding of the role of toxin in pathogenicity and disease development. he role of CWDEs is well established in virulence of the fungus and it may also aggravate the disease severity in the presence of phaseolinone. However, the interaction of CWDEs and the phytotoxic metabolites in the initiation and development of disease symptoms remains to be diagnosed in detail to understand the mechanism and their role in pathogenesis. his will substantially accelerate the ongoing eforts to understand the complex susceptibility determinants in the host. Further research on these enzymes will open new avenues for planning plant-protection strategies. Acknowledgements he authors acknowledge ICAR, New Delhi, India for providing Senior Research Fellowship for doctorate studies and Canadian Government for providing Canadian Commonwealth Scholarship (CCSP, 2010-11) to Miss Surinder Kaur. he views or opinions expressed in this article are those of the authors. Declaration of interest he authors declare no declarations of interest. References Abawi GS, Pastor-Corrales MA. (1990). Root rot of beans in Latin America and Africa: Diagnosis, research methodologies and management strategies. 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