surya
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surya

Course Number: BIOL 4500, Fall 2009

College/University: Laurentian

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Research Signpost 37/661 (2), Fort P.O., Trivandrum-695 023, Kerala, India Advances in Plant Disease Management, 2003: 345-371 ISBN: 81-7736-191-0 Editors: Hung-Chang Huang and Surya N. Acharya 17 Senior Author Breeding alfalfa for resistance to verticillium wilt: A sound strategy Surya N. Acharya and Hung-Chang Huang Agriculture & Agrifood Canada Research Centre, P.O. Box 3000, Lethbridge, Alberta Canada...

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Signpost Research 37/661 (2), Fort P.O., Trivandrum-695 023, Kerala, India Advances in Plant Disease Management, 2003: 345-371 ISBN: 81-7736-191-0 Editors: Hung-Chang Huang and Surya N. Acharya 17 Senior Author Breeding alfalfa for resistance to verticillium wilt: A sound strategy Surya N. Acharya and Hung-Chang Huang Agriculture & Agrifood Canada Research Centre, P.O. Box 3000, Lethbridge, Alberta Canada T1J 4B1 Dr. Surya N. Acharya has been a research scientist at the Agriculture and Agri-Food Canada, since 1989. He has been involved in genetic improvement of traditional and non-traditional forage crops. To date he has developed and released two alfalfa, nine native grass and one each of cicer milkvetch, orchardgrass and perennial cereal rye cultivars for commercial production in western Canada. His research interests include genetic improvement in resistance to diseases, acid and saline soil conditions as well as improvement in winterhardiness and grazing tolerance and combining these with improved biomass yield and nutritional quality of forage crops. Abstract Verticillium wilt of alfalfa, caused by Verticillium albo-atrum Reinke & Berthold, is a devastating disease that can cause millions of dollars in losses to alfalfa producers in cooler regions of the world. The disease can spread to disease free fields through infected debris, insects, irrigation water, wind and harvesting equipment. The fungus is host specific and plants inoculated with isolates from alfalfa produce a characteristic wilt symptom. All cultivars, including those that are considered susceptible, have some plants which will not develop symptoms when subjected Correspondence/Reprint request: Dr. Surya N. Acharya, Agriculture & Agrifood Canada Research Centre, P.O. Box 3000 Lethbridge, Alberta, Canada T1J 4B1. E-mail: acharya@agr.gc.ca 346 Surya N. Acharya & Hung-Chang Huang to artificial inoculation. Genetic analyses and research on resistance mechanism in plants have indicated that verticillium wilt resistance in alfalfa is controlled by multiple genes. Selections for verticillium wilt resistance in alfalfa have been successful worldwide, including the breeding program at the Lethbridge Research Centre, Alberta, Canada. The success of the Lethbridge program may have been due to simultaneous selection for plants with disease resistance, high yielding ability and adaptation to western Canada growing conditions. This review elaborates on the above aspects of this important disease with special emphases on epidemiology, genetics and source of resistance, the method used for breeding resistant cultivars and economic impact of growing verticillium wilt resistant cultivars of alfalfa. Introduction Alfalfa (Medicago sativa L.) has gained importance over time as a forage crop and is considered an integral part of livestock production in many countries, including USA and Canada. Alfalfa, often referred to as queen of forages, is the basic component in feeding programs for dairy and beef cattle. Processed alfalfa, cubes and pellets, are used for feeding large and small animals, while alfalfa sprouts are now a popular addition to human food. Verticillium wilt, caused by a relatively host-specific strain of Verticillium albo-atrum Reinke & Berthold, is a devastating disease of alfalfa in Europe and North America. This disease affects alfalfa producers through reduction in yield and quality of alfalfa hay and seeds as well as the life of the alfalfa stand. This disease also has negative economic impact on associated industries such as alfalfa processing, cattle, dairy as well as leafcutter bee industries. This review contains the history of verticillium wilt in major alfalfa growing regions of the world, economic importance of the disease, epidemiology of the pathogen and factors affecting spread of the disease and the control of the disease through alfalfa breeding. Special emphases are given to the screening techniques used for selecting verticillium wilt resistant plants, the breeding for high levels of resistance in alfalfa, performance of resistant and susceptible cultivars in the field and the economic impact of growing verticillium wilt resistant cultivars. Occurrence of verticillium wilt of alfalfa Verticillium wilt of alfalfa was first reported in Sweden in 1918 (Hedlund, 1923). By the mid 1940s, it was found widely in the cooler regions of continental Europe (Kreitlow, 1962) and in the early 1950s in Great Britain (Atkinson, 1981). By the 1950s, verticillium wilt was beginning to be recognized as a major disease of alfalfa in continental Europe (Isaac, 1957). Verticillium wilt of alfalfa in North America is considered a humble immigrant brought over from the Old World (Heale, 1985). Incidence of verticillium wilt in North America was first reported in 1962 when V. alboatrum was isolated from alfalfa in the Experimental Farm plots of Normandin, Quebec, Canada (Aube and Sackston, 1964). About the same time, the disease was found in research plots at the University of British Columbia, Canada. Although these early occurrences did not result in wide spread disease incidences in North America (Kreitlow, 1962), it gave warning of the potential threat of this disease to the continent. The concern was important because of cotton-alfalfa rotation in New Mexico and the apparent susceptibility of cotton to what was then called a microsclerotial form of Alfalfa resistance to verticillium wilt 347 V. albo-atrum (now known as Verticillium dahliae Kleb.). Although V. dahliae was reported to attack alfalfa (Isaac 1957), Smith (1961) found that alfalfa cultivars were not susceptible to the V. dahliae from cotton. Because of the destructive effect of verticillium wilt on production of alfalfa hay and seed and the transmission of this disease through infected alfalfa seeds and accompanying debris, quarantine measures for the seeds imported from Europe were instituted in USA and Canada (Christen and Peaden, 1981). This meant that all the imported seed would be treated with recommended fungicides before they were allowed into the country. In spite of these measures, verticillium wilt of alfalfa was again found in 1976 in Washington State (Christen and Peaden, 1981) and in the neighbouring Canadian Province, British Columbia in 1977 (Sheppard, 1979). Christen and Peaden (1981) estimated that the disease was probably introduced in 1972 or 1973 to Washington State. Although early occurrences of verticillium wilt were known, it was not until 1976 that consistent reports of the disease in North America appeared (Huang and Atkinson, 1982; Heale, 1985; Arny and Grau, 1985; Howard et al., 1991). By late 1970s, verticillium wilt had become wide spread in North America (Heale et al., 1979) and the need for control measures was considered urgent. At the time, verticillium wilt of alfalfa was most prevalent in cooler regions of North America and was thought to be restricted to areas north of 40o latitude (Arny and Grau, 1985). Further disease surveys found the disease to be distributed more widely even in warmer regions (Heale et al., 1979) including southern California (Erwin et al., 1989; Howell and Erwin, 1995). In Japan, verticillium wilt of alfalfa was first detected in 1981 in Sorachi, Hokkaido (Sato, 1994). An extensive survey of the Hokkaido area revealed that the disease was present all over the island causing severe damage to the crop. This report also indicated that all the alfalfa cultivars grown in the area at that time were susceptible to the disease (Sato, 1994). From 1950s to 1980s, research in Europe concentrated on the causal organism (V. albo-atrum.), disease symptoms, field management of the disease, transmission via seed and weeds, yield losses and breeding for resistance (Isaac, 1957; Isaac and Lloyd, 1959; Isaac and Heale, 1961; Heale and Issac, 1963; Panton, 1965; 1967b; Aubury and Rogers, 1969; Lundin and Jonsson, 1975; Gondran, 1978). Since 1980s, this disease has been recognized as a serious problem of alfalfa in North America and research efforts in Europe and North America have been directed towards understanding the disease, its transmission and subsequently control through incorporation of genetic resistance into new cultivars. Econonic importance of verticillium wilt of alfalfa Verticillium wilt of alfalfa can cause serious economic losses to hay producers in several ways. The first is the loss due to lower production caused by thinning of the stand over time and the second is the cost associated with reduced stand life. The wilt infected plants die the second or third year after planting, reducing the yield below economically productive levels and forcing early replanting. A cause not taken into account in the earlier economic analyses, is the loss associated with reduction in hay price when weeds are mixed with the hay. As the alfalfa stand thins, weeds invade the stand and consequently hay produced in these fields is sold at a discounted price compared to clean alfalfa hay (Smith et al., 1995). Alfalfa seed production can also be adversely affected by verticillium wilt (Atkinson, 1981). Although there is no report on 348 Surya N. Acharya & Hung-Chang Huang exact loss of seed due to this disease, it is well known that the pathogens borne externally and internally in the seeds are responsible for transmission of the disease to uninfected fields. Assessments of the damage to alfalfa due to verticillium wilt have been reported in Canada (Aube and Sackston, 1964; Huang et al., 1994), USA (Page et al., 1992), Czechoslovakia (Kudela and Malik, 1974), Germany (Steuckardt et al., 1971), Denmark (Nielsen and Andreasen, 1970), England (Aubury and Rogers, 1969), in France (Gondran, 1977), Italy (Ranella et al., 1969), Hungary (Szoko, 1966), Sweden (Lundin, 1969) and Japan (Sato, 1994). A study in France estimated that verticillium wilt of alfalfa caused a loss of about 1 Mg of dry matter per ha per year (Gondran, 1984). In 1980s, there were about 400,000 ha of alfalfa cultivated in the area to the north of DijonPoitiers line, resulting in substantial loss due to the disease. In the Hokkaido area of Japan, the disease caused a hay yield loss of about 33% in susceptible cultivars while the yields in resistant cultivars were not affected (Sato, 1994). In Wyoming, USA, alfalfa yield loss was estimated by comparing resistant and susceptible cultivars grown in the presence of verticillium wilt. Using data from a field test conducted during 1984-87 and survey of the area infested with V. albo-atrum, Page et al. (1992) estimated the economic impact of the wilt disease on alfalfa hay production in the state. Resistant cultivars had less disease, higher yield and higher plant stands than susceptible cultivars. Average annual yield loss attributed to verticillium wilt was 0.88 Mg ha-1. Approximately 32,877 ha of alfalfa hay grown under irrigation in Wyoming were infested with V. albo-atrum. Out of that an estimated 27,946 ha was planted to susceptible cultivars. With an average hay price of $70.46 Mg-1, the annual loss in hay production in the state of Wyoming was estimated to be $1.7 million per year. From a study in Ithaca, New York, Viands et al. (1992) reported mean yield advantage due to verticillium wilt resistance in the highly resistant cultivar Oneida VR to be 1.2 Mg ha-1 (valued at $109 ha-1 at $94 Mg-1 hay) in the second production year and 2.87 Mg ha-1 (valued at $270 ha-1) in the third year compared with the most susceptible cultivar Saranac AR. These estimates did not take into account reduced value for weed infested hay in susceptible cultivars. Using data from a 7-year study conducted in an irrigated field naturally infested with V. albo-atrum in southern Alberta, Canada (Huang et al., 1994), economic benefits of growing resistant cultivars were estimated (Smith et al., 1995). This analysis used reduction in forage yield over the duration of the study, stand replacement cost and reduced value of hay due to weed infestation in the calculations. The yearly benefit of alfalfa cultivars with high resistance to verticillium wilt over moderate resistance was estimated at $21 ha-1 and over low resistance was $44 ha-1. This translated into a potential yearly benefit of $2.2 million to producers in western Canada from growing only resistant alfalfa cultivars (Smith et al., 1995). Epidemiology of verticillium wilt of alfalfa Verticillium wilt of alfalfa is caused by a special strain of V. albo-atrum (Fig. 1). Field symptoms of verticillium wilt are distinct in the early bud stage of plant growth and they consist of V-shaped pinkish orange brown necrotic patches on the leaflets (Fig. 1 insert). Leaflets on severely affected shoots are usually necrotic and twisted. Diseased stems remain green and erect (Fig. 1) even after all leaves have wilted. New shoots on Alfalfa resistance to verticillium wilt 349 Figure 1. Verticillium wilt infected plant with erect stems and wilted leaves. Note V-shaped lesions on the leaflets (insert). Figure 2. A conidiophore of Verticillium albo-atrum with whorled branches. infected plants appear normal at first but show typical symptoms as they approach physiological maturity (Christen and Peaden, 1981). Symptoms of the disease can be produced within a short time through artificial inoculation under controlled environments of 15 27 oC (Huang and Hanna, 1991). High soil moisture is conducive 350 Surya N. Acharya & Hung-Chang Huang to the development of verticillium wilt of alfalfa, as the disease is found primarily in irrigated fields and in areas with high humidity (Arny and Grau, 1985; Howard et al., 1991). Diseased plants are often stunted (Fig. 1). All shoot may be affected, but more frequently only one or two stems show symptoms. Initially few wilting plants are observed but as the disease spreads, the number of wilted plants become more numerous and the whole field turns into patches of infested plants. These patches are more common in hay fields than in seed fields (Balliette et al., 1993). Other diseases, such as bacterial wilt and phytophthora root rot, may show similar symptoms and therefore, isolation for V. albo-atrum (Fig. 2) from infected leaflets or stems is required for positive identification of verticillium wilt of alfalfa. Under severe winter conditions of northern latitudes the infected plants may die sooner due to added stress of winter injury and the dead plants allow weedy species to invade the open areas (Fig. 3). Figure 3. A 7-year old alfalfa test (1986 - 1993) at Lethbridge, Alberta showing dandelion (weed) invasion in the plots. Green plots (uninfested) are cultivars having resistance to verticillium wilt and yellow plots (heavily infested) are susceptible cultivars. V. albo-atrum is generally introduced into alfalfa fields by infected seeds or through harvest equipment used in infested fields, windborne spores from adjacent fields or through infested hay (Grau et al., 1982) and it may over winter on diseased stubbles in the field (Huang et al., 1983; 1994). In 1980s, several insect species were found to be vectors of verticillium wilt of alfalfa and they play a major role in the transmission of the disease to clean fields (Huang et al., 1983; 1986). Disease transmission through infected seeds and debris Isaac (1957) first showed that V. albo-atrum could be distributed around Europe in contaminated alfalfa material (fragments of stem, pedicels or pods) collected during harvesting of the seed. Sheppard and Needham (1980) isolated V. albo-atrum from a number of alfalfa seed lots of diverse origin. In most cases they found that the organism was carried with the seed lot on pieces of plant debris or on the surface of the seed. The spores formed on the surface of the seeds during damp storage will remain viable and be distributed with the seed. The propagules in the debris occur primarily in the form of dark resting mycelium (Isaac and Heale, 1961), which under moist conditions gives rise to conidia. In Japan, Sato (1994) found that V. albo-atrum was introduced to the field through the imported infected seeds and it survived in plant residues contaminated during the harvesting operations. Use of protective seed dressing fungicide such as Alfalfa resistance to verticillium wilt 351 Thiram was recommended for preventing contamination of alfalfa seeds and debris by V. albo-atrum. Christen (1983) reported that surface sterilization (using sodium hypochlorite 5.25 % (Clorox), 70 % ethanol, (1:9; v:v) mixture and 0.1 % wetting agent for 45 sec) of 10, 000 seeds from alfalfa crops grown in the Columbia basin of Washington, completely eliminated the 2 % of seeds infested with the externally borne V. albo-atrum. Basu (1987) demonstrated that the pathogen within the host stem tissue (stem cuttings) remained viable for over 3 years, suggesting that the contaminated alfalfa stems may serve as a continual source of infection in the field. More importantly, the pathogen in this case is inside the tissue and so treatments with fungicides, such as, Thiram will not completely eliminate the pathogen which will be able to transmit the disease to healthy plants for a long time. A small proportion of the seeds carried the pathogen internally (Isaac and Heale, 1961; Sheppard and Needham, 1980) for which sanitation or fungicide treatments of the seed are not effective in controlling spread of the disease. Christen (1982) reported internal infection within and between osteosclerid cells of the outer integument of the seed coat. Internal seedborne infection was as high as 25% in the small seeds produced on plants experimentally inoculated by stem-injection of V. albo-atrum just below each raceme within 2 weeks of pollination. Infection occurred less frequently in larger seeds. A small proportion of infected seeds (3 infected seeds from 40 racemes) were also collected from naturally infected plants from an experimental plot. From this observation, it was concluded that such seed coat infection was unlikely to be totally eliminated by protective seed dressings (Thiram) as was recommended in earlier years. The best means of controlling this disease is, therefore, through the use of resistant cultivars. Disease transmission through insects Studies in commercial fields of alfalfa indicated that nine species of alfalfa pests and six species of predatory insects served as agents for dispersing conidia of V. albo-atrum. Sucking insects such as the pea aphid (Acyrthosiphon pisum Harris) (Huang et al., 1983) were proven effective vectors for V. albo-atrum spore transmission from infected to uninfected alfalfa plants. Laboratory plating of aphids collected from alfalfa fields indicated that spores of V. albo-atrum were carried as surface contaminants, most commonly on the legs and antennae of aphids and spores borne on this insect can induce disease symptoms on healthy, alfalfa plants. Field-collected chewing insects such as alfalfa weevils (Hyper postica (Gyllenhal)), contaminated with spores of V. albo-atrum, were able to cause verticillium wilt when caged on healthy alfalfa (Harper and Huang, 1984). Feces of leaf-chewing insects such as grasshoppers and alfalfa weevils were also instrumental in spreading the pathogen (Huang and Harper, 1985). Even beneficial insects such as leafcutter bees (Megachile rotundata (Fabricius), principal pollinator for alfalfa seed production in Canada, were responsible for transmission of V. albo-atrum. Alfalfa leaf pieces cut by leafcutter bees for use in the construction of hive cells were frequently contaminated with V. albo-atrum (Huang and Richards, 1983). A small percentage of leaf pieces originating from infected leaves could contaminate the entire hive. Other studies on the mechanisms of V. albo-atrum spread in alfalfa indicated that alfalfa pollen on the stigma of alfalfa flowers (Huang et al., 1985) or on agar media 352 Surya N. Acharya & Hung-Chang Huang (Huang and Kokko, 1985) are susceptible to infection by the pathogen. Therefore, the leafcutter bees carrying alfalfa pollen are likely an important agent for dissemination of V. albo-atrum. By nature, these bees trip alfalfa flowers and in the process gather pollen and nectar as well as cut pieces of alfalfa leaves to make bee cells. Bees use leaf pieces from both healthy and diseased plants to construct brood cells (Huang et al., 1986). In this field study 30% of the bees collected were contaminated with the pathogen and conidia of V. albo-atrum were present in most parts of their body. On some bees, conidia of V. albo-atrum had germinated and produced hyphae for infection of pollen grains carried by the bees. It was possible to isolate V. albo-atrum from stigma and styles of healthy looking plants confirming that leafcutter bees are an important agent for dissemination of this pathogen. Alfalfa pollens were susceptible to in vitro infection by V. albo-atrum. The pathogen infected pollen grains can be carried by leafcutter bees to non-infected flowers and healthy plants where they can start a secondary infection (Huang and Kokko, 1985). These observations suggest a strong possibility that V. alboatrum can be transmitted to alfalfa seeds through the process of bee pollination (Huang et al., 1985; 1986). Disease transmission by wind, water and farm implement The resting mycelium of V. albo-atrum in disease alfalfa tissues can produce conidiophores (Fig. 2) and conidia under cool, moist conditions (Huang et al., 1983). These conidia can become airborne and have been trapped over alfalfa fields (Isaac, 1957). Conidia dispersed by activity of machinery and by air currents could land on cut alfalfa stems, resulting in infection. Water has also been reported to be a means by which Verticillium spp. are spread locally as well as long distance (Howard, 1985). Easton et al. (1969) detected a large number of viable propagules of V. albo-atrum per L of waste water in an irrigation settling pond located 1.6 km from the diseased field. In Alberta, Howard (1985) found large amounts of wilt-infected debris on haying machinery, i.e. balers, mowers etc. used for harvesting diseased fields. Pathogenicity of Verticillium albo-atrum strains Only isolates of V. albo-atrum from alfalfa are pathogenic to alfalfa (Isaac and Lloyd, 1959; Heale and Isaac, 1963). A variety of weeds in diseased alfalfa fields can harbour the pathogen without showing symptoms other than stunting (Heale and Isaac, 1963). Various clovers (Trifolium spp.) were found to be resistant to V. albo-atrum; while other legumes such as lupin, pea, red clover, sainfoin, soybean, sweet clover and white clover were found to harbour the alfalfa strain of V. albo-atrum without suffering noticeable damage (Yorston, 1982). The fact that the disease was not found in southern France or in Mediterranean countries during early years indicated that the disease pathogen proliferates mostly under cool temperature conditions. However, this myth was dispelled when it was found in southern California (Erwin and Khan, 1988; Erwin et al., 1989) and the pathogen remained viable for 32 months in the infected hay bales exposed to air temperatures as high as 40 oC during summer months (Howell and Erwin, 1995). Christen and French (1982) indicated that the alfalfa strains of V. albo-atrum may be distinguished from strains of Verticillium spp. from other hosts by higher temperature optima for radial growth on agar media. Isaac (1949) found a temperature optimum of 20 22.5 oC for four isolates of V. albo-atrum while Domsch et al. (1980) found 21 oC (20 - Alfalfa resistance to verticillium wilt o 353 24 C range) to be optimal for other isolates of this pathogen in four independent studies. These results implied that the alfalfa strains of V. albo-atrum can spread into relatively warmer regions due to its adaptation for warmer temperature conditions. It is therefore possible that the disease could spread into areas where previously only V. dahliae caused damaging wilt diseases in major crops (Heale, 1985). Christen et al. (1983) showed that temperature affected symptom expression and differential alfalfa cultivar reaction to V. albo-atrum. Delwiche et al. (1983) found greater symptom expression at 20 to 24 oC than at 28 to 32 oC and low light intensities favoured symptom expression. Understanding temperature optima for V. albo-atrum has helped in the development of indoor screening techniques for selecting resistant plants within open pollinated populations of alfalfa. Peaden (1974) outlined standardized test procedures for assessing verticillium wilt resistance in alfalfa where environmental parameters and the density of inoculum were specified and kept constant. Comparison of virulence among alfalfa isolates of V. albo-atrum from Oregon, USA, U.K. and France, on cultivars DuPuits, Pheonix, Sabilt and Maris Kabul revealed higher virulence for USA isolates than those from Europe. Differences in virulence among alfalfa isolates have also been suggested by Muller (1969), Renella et al. (1969) and Gondran (1981). In Hokkaido, Japan, pre-inoculation of alfalfa plants with the potato strain of V. albo-atrum resulted in a decrease in severity of verticillium wilt in alfalfa (Sato, 1994). In an investigation conducted simultaneously at Queen Elizabeth College (QEC), London, U.K. and the Irrigated Agriculture Research and Extension, Washington State University, Prosser, WA, USA, North American and European isolates of V. albo-atrum from alfalfa were tested against USA and European alfalfa cultivars. At QEC fluctuating 17-30 oC (night/day) and 12-21 oC temperature regimes were used, while at Prosser a constant regime (27 1 oC) was used. Disease severity was found to be significantly greater at the higher temperatures at both centers (Christen et al., 1983). Relative ranking of the cultivars was similar throughout, with Maris Kabul being most resistant (80 % resistant plants). In this study, small differences in virulence were noticed among the isolates of V. albo-atrum suggesting common origin of these isolates of V. albo-atrum (Christen et al., 1983). Breeding alfalfa for resistance to verticillium wilt Source of resistance An interesting observation with regard to verticillium wilt resistant alfalfa is that all cultivars including those that are considered susceptible have some plants that will not develop symptoms when subjected to artificial inoculation (S. N. Acharya and H. C. Huang, unpublished). Resistant cultivars, however, show a higher proportion of symptomless plants compared to susceptible cultivars. This and the fact that some plants develop verticillium wilt symptoms many months after inoculation indicate that symptomlessness may be due to tolerance and not true resistance. Such observations also lead some to infer that verticillium wilt resistance in alfalfa is controlled by a multigenic system (Paton, 1965). Recurrent selection for resistance within commercial European cultivars of alfalfa has produced cv. Vertus, which exhibits reduced colonization and a degree of tolerance to verticillium wilt. One diploid wild alfalfa Medicago hemicycla L., was found to have 354 Surya N. Acharya & Hung-Chang Huang marked limitation of colonization by the verticillium wilt pathogen (Heale, 1962). Crosses between chromosome doubled M. hemicycla and M. sativa lines followed by repeated backcrossing to M. sativa and selection led to the development of cv. Maris Kabul. This cultivar released in 1970 is known to have extremely low colonization by V. albo-atrum (Heale, 1985) and is used extensively as a source of resistance for this disease. Another moderately resistant, low yielding wild alfalfa M. gaetula L. was crossed to Saranac and Canadian Poly for development of Maris Phoenix (Heale, 1985). It was released in U.K. in 1970 but has not been widely used because of its poor yielding ability and relatively low resistance to verticillium wilt (Busch and Smith, 1981). One other main source of resistance for this disease is Vertus, a cultivar developed by Lundin and Jonsson (1975) in Sweden by selecting resistant plants from a commercial cultivar. Resistant plants have also been found in M. sativa x M. falcata hybrids and from many cultivars grown commercially for their higher yield and adaptation (Elgin et al., 1988). Although recent progress in cell and tissue culture techniques for alfalfa is leading to the selection of novel sources of wilt resistance, most of the resistant cultivars developed to date have used Vertus or Maris Kabul directly or indirectly as source of resistance. Mechanisms of resistance Mechanisms of verticillium wilt resistance in alfalfa have been investigated and discussed in a review (Heale, 1985). At the plant tissue level, Heale (1985) observed a marked discontinuous upward colonization in the roots of two resistant cultivars Maris Kabul and Vertus. The colonization was particularly limited in Maris Kabul. Roots of both cultivars were found to contain occluding material in 10% of the root xylem vessels which was absent from non-resistant plants. In resistant plants, xylem parenchyma cells often showed an increase in mitochondrial profiles, vesicular content and endoplasmic reticulum, with an increase in nuclear/nucleolar volumes, as well as cell wall (Heale, 1985). Other associated xylem parenchyma cells in contact with colonized vessels underwent degenerative changes as is seen in a hypersensitive reaction (Robb et al., 1982). In resistant cultivars, many vessels, with or without occlusions, were lined with electron-dense material and were blistered in some cases. Pit membranes showed various stages of disintegration and blocking with the electron-dense material. Heale (1985) suggested that recognition events may have caused such reaction in resistant cultivars when the pathogen or its soluble products interacted with the adjacent living xylem parenchyma cells. As part of a hypersensitive response, the metabolic activity of the parenchyma cells appears to be elevated and secretion of material occurs via the pits that may contribute to the occlusions. Phytoalexins were postulated to accumulate within adjacent V. albo-atrum affected vessels, which are subject to water-proofing by the electron-dense lining material (Heale, 1985). Leaf tissues of DuPuits, a susceptible cultivar, accumulated phytoalexins such as medicarpin and sativan rapidly in response to a non-pathogenic race of V. albo-atrum. The stems (but not leaves) of resistant Maris Kabul accumulated these phytoalexins more rapidly than DuPuits (Heale, 1985). There may also be a role for inhibitors in differential cultivar resistance to verticillium wilt in alfalfa. Saponins of resistant wild alfalfa M. hemicycla showed a high fungistatic activity while M. sativa did not (Jurzysta and Nowacki, 1979). Manninger et Alfalfa resistance to verticillium wilt 355 al. (1978) also observed increasing inhibition of V. albo-atrum at high saponin levels but, did not observe correlation between wilt resistance and overall saponin content of alfalfa cultivars. At the whole plant level, V. albo-atrum disrupts normal plant functions in several ways, including disruption of xylem vessel differentiation (Pennypacker and Leath, 1986) and impediment of water movement through the host, suggesting different genotypes may have different mechanisms of resistance. From a series of studies, Viands (1985) concluded that at least two genetic mechanisms may be controlling resistance to verticillium wilt in alfalfa. Pennypacker and Leath (1993) observed two distinct histological reactions to V. albo-atrum from two resistant alfalfa cultivars, further strengthening Viands (1985) contention of two resistance mechanisms controlled by separate genetic systems. Yu et al. (1993) reported low correlation coefficients between different methods of inoculation of stems and leaves indicating the complexity of the interaction between the pathogen and the host. They, therefore, suggested that one method of inoculating the stems may not mimic all of the effects that a fungal infection has on a plant and consequently, no specific screening method would screen for all types of resistance. Genetics of resistance Genetic studies of disease resistance in alfalfa are more complicated than in other field crops. First of all, alfalfa (M. sativa) is an autotetraploid species having four loci for each gene. In this case, the usual diploid genetic ratios are not observed. In addition, involvement of more than one locus in determining resistance can make genetic ratios extremely complex. Secondly, alfalfa is a cross-pollinated crop with some level of self incompatibility. This crop is totally dependant on insects for pollination. Each plant can be unique in genotypic constitution and most plants are heterozygous for most traits. Therefore, plants in a population may not be all resistant (Christie et al., 1985). Perennial nature of the plants and slow development of the disease symptoms makes genetic studies for verticillium wilt resistance in alfalfa further complicated. Resistance to V. albo-atrum involves vascular coating materials, antifungal compounds, gel formation and tylosis formation (Newcombe et al., 1990). In spite of the difficulties in studying genetics of resistance, initial reports on the inheritance of resistance showed that resistance was conditioned by a multigenic system with predominantly additive genes (Fyfe, 1964; Panton, 1967a; b). Steuckardt et al. (1971) found that the resistance of alfalfa to V. albo-atrum is expressed by a polygenic system of genes. Guy and Genier (1974) observed slow improvement in the level of resistance through many cycles of mass and intra-population selection. They attributed the slow progress to the polygenic nature of inheritance for the trait. Moller and Andreasen (1975) suggest that several alleles and loci are involved in conferring resistance. Fyfe (1964) explained differences in field resistance on the basis of combining ability effects, while, Panton (1967b) found significant general (GCA) and specific combining ability (SCA) effects for resistance. Using GCA and SCA estimates, Miller and Christie (1991) suggested that additive genetic variance will provide limited improvement of resistance levels and nonadditive genetic variance will not improve resistance levels much if one was to use Vertus as the source of resistance to verticillium wilt. 356 Surya N. Acharya & Hung-Chang Huang Gondran (1984) showed that the susceptible plants of alfalfa are completely colonized by V. albo-atrum, when only the stems cut by infected scissors are invaded in the resistant plants but their regrowths are not colonized. Christie et al. (1985) suggested that stem colonization data would be useful in the identification of alfalfa plants resistant to verticillium wilt and so help genetic studies for this trait. Using this technique, Papadopoulos (1987) investigated the extent and rate of stem colonization for eight breeding lines from two resistant cultivars, Maris Kabul and Vertus, and two susceptible cultivars, Apollo and Banner. A significant correlation (r = 0.79) between average index of stem colonization and foliar disease severity index was observed. Later a significant correlation between stem colonization index (determined under a controlled environment of 18 oC and 16 h daylength) and foliar disease severity in the field was observed (Papadopoulos et al., 1989) indicating the usefulness of this technique in identifying resistant plants. Viands (1985) studied genetics of resistance to verticillium wilt in Maris Kabul and Vertus. His quantitative genetic analyses indicated that resistance in each cultivar was controlled by additive genes, but non-additive effects were detected within Maris Kabul. In this study, qualitative genetic analysis supported the additive gene hypothesis in Vertus, but segregation ratios within Maris Kabul suggested a single dominant gene along with additive genes conferring resistance. Toth and Bakheit (1983) reported multigenic control for resistance and some genes with additive effect; while, Papadopoulos (1987) found that variations in resistance was due primarily to additive gene effect, although there was some evidence of non-additive gene action. Christie et al. (1985) selected alfalfa plants resistant to V. albo-atrum from North American and European cultivars. These selected plants were then inter-crossed to produce synthetics and compared with synthetics made from random plants of adapted cultivars. The Syn1 generation of these synthetics varied in level of resistance (range 3 89%), suggesting that resistant cultivars can be produced through simple phenotypic selection. However, the wide range in resistance observed indicated that resulting cultivars may vary substantially in their ability to resist infection by V. albo-atrum. A quantitative genetic analysis using self-pollinated and test-crossed progenies of six selected plants did not reveal a conclusive segregation pattern for verticillium wilt resistance which was attributed to improper classification of the selected plants. Christie et al. (1985) evaluated levels of phenolic compounds and of stem colonization by the pathogen and found them to be more suitable for distinguishing wilt-resistant and wiltsusceptible cultivars than simple phenotypic selection. In a test to determine efficiency of controlled environmental methods to evaluate verticillium wilt resistance in the field, Papadopoulos et al. (1989) found that the evaluations of cultivar resistance to the disease were in agreement with the cultivars' reaction in the field following natural infection. Field disease ratings were positively correlated with growth chamber disease ratings and negatively correlated with herbage dry matter yield. Twenty to 56% of the variation in herbage yield among cultivars in the V. albo-atrum infested field was attributed to the influence of disease. Foliar disease symptoms appeared in the field about 14 months after seeding; while, differences in reductions in healthy plants and herbage dry matter yield were detected about 28 months after seeding. Therefore, indoor evaluation was considered a rapid method of separating resistant and susceptible cultivars. These authors estimated that alfalfa cultivars should Alfalfa resistance to verticillium wilt 357 possess levels of resistance comparable to that of the check cultivar Vertus to avoid yield losses due to this disease. Busch and Christie (1982), on the other hand, suggested a target minimum of 60 % resistant plants for selected Canadian alfalfa cultivars in their breeding program. Several lines of evidence indicate the polygenic nature of resistance to V. alboatrum: (a) the improved cultivars have remained durable; (b) transgressive segregation occurs (Panton, 1967a); and (c) there is considerable variation in levels of resistance of individual plants in a population grown from a particular seed lot. The latter phenomenon is due to the out-breeding and uncontrolled pollination used in the production of seed in this autotetraploid crop which leads to genotypic variation in the F1 and subsequent generations (Little, 1958). Kehr et al. (1972) have questioned whether any alfalfa cultivar can be stated to be resistant because of this marked heterogeneity. Whitney et al. (1972), studying protein differences possibly related to resistance, selected resistant individual plants grown from three heterogeneous seed samples of resistant M. sativa selections. Even though the heritability of resistance was low, recurrent phenotypic selection for resistance was effective (Elgin et al., 1988). LatundeDada and Lucas (1982) reported on the heterogeneous nature of resistance in five commercial alfalfa cultivars, including Maris Kabul, Sabilt and Vertus, and pointed out that each cultivar contains only a proportion of resistant phenotypes. It was also interesting to note that selection for forage quality did not affect levels of disease resistance, although it may have reduced plant vigour (Fonseca et al., 1999). Screening techniques An effective verticillium wilt screening procedure for alfalfa should be simple, rapid and provide a clear distinction between susceptible and tolerant (resistant) genotypes (Durbin 1981; Huang and Hanna, 1991). More importantly, it should be applicable to genotypes of diverse origin to be useful for screening plants for cultivar improvement. Many methods used for the purpose appear to satisfy these criteria. Some methods are non-destructive and can take only a few days while others take longer. (i) Stem cuttings Yu et al. (1993) compared three screening tests for resistance to verticillium wilt in alfalfa using stem cuttings. The three methods included stem infusion with a fungal culture filtrate, leaf injection with spore inoculum and stem infusion with spore inoculums. The disease severity indices, determined by the three tests, were very similar on average and were significantly correlated for a population of 142 plants regenerated from tissue culture. The disease severity indices, determined by the leaf injection with a spore inoculum test were also significantly correlated with the disease severity indices determined by the North American Standard test (Peaden, 1974) in a population of 20 Vertus alfalfa plants. The results suggest that the assay using stem cuttings are effective for determining resistance to verticillium wilt in alfalfa. These tests are non-destructive, the original plants are not infected by the procedure, the tests can be replicated and the results are available within 7-8 days. In contrast, disease symptoms in the North American Standard test are not evaluated until 5 weeks after inoculation and can only be done once on a particular plant. Therefore, Yu et al. (1993) suggested that stem infusion with a spore inoculum or with a fungal culture should filtrate find application in alfalfa 358 Surya N. Acharya & Hung-Chang Huang breeding. The stem cutting and infusion methods can be used to check symptomless plants from conventional screening procedures for escapes. (ii) In-vitro methods Conventional methods for breeding alfalfa resistant to verticillium wilt are complex because alfalfa is an autotetraploid crop and suffers from severe inbreeding depression (Christie et al., 1985) if self-pollinated. There was considerable interest in incorporating tissue culture techniques into plant improvement programs because they can simplify the production of novel genetic variants and can be used to screen large populations in a small space under closely controlled conditions. A protocol used to select disease-resistant lines in vitro is to grow callus or suspension culture cells in the presence of a fungal culture filtrate containing toxic metabolites produced by the pathogen (Arcieni et al., 1987; Daub, 1986; Dixon, 1980; Ingram and MacDonald, 1986; Wenzel, 1985). The toxic component may be hostspecific and has shown to play a critical role in pathogenesis (Gengenbach and Green, 1975), or may be non-specific toxins shown to be important for disease development (Mitchell, 1984). Both Panton (1965; 1967b) and Michail and Carr (1966) showed that toxin-containing culture filtrates of the pathogen could be used in screening procedures, since resistant selections were less affected than susceptible materials. In vitro disease resistance studies with alfalfa callus and fungal pathogens were carried out by Miller and Maxwell (1984). The in vitro alfalfa-Verticillium interaction has been characterized by Latunde-Dada and Lucas (1985; 1986), who utilized alfalfa callus and live fungus to investigate the role of phytoalexins in the resistant response. Frame et al. (1991) found a filtrate from mycelial cultures of V. albo-atrum to inhibit the cell growth and reduce the viability of alfalfa suspension cultures. The toxicity of the filtrate was enhanced at pH 7.5 relative to pH 5.5. A similar filtrate preparation from V. dahliae, which did not cause disease symptoms in alfalfa, was also toxic to the alfalfa suspension culture cells. Plants regenerated from cells resistant to the V. albo-atrum filtrate from two genotypes had significantly lower average disease severity indices than plants regenerated from control cultures and the donor plants. Also, the average disease severity indices of plants regenerated from filtrate-treated cultures of one genotype decreased from cycle to cycle, but remained the same from plants regenerated from control cultures. This indicates that the fungal culture filtrate technique can be used for examining non-specific host-pathogen interactions and to obtain plants with increased resistance to verticillium wilt in alfalfa. The prospect of using somaclonal variation to generate novel sources of resistance to verticillium wilt is an exciting one, and some progress in improvement has been reported for alfalfa. Latunde-Dada and Lucas (1983) showed that variant protoclones derived from mesophyll protoplasts of cv. Europe were highly tolerant to the V. albo-atrum alfalfa strain although this is probably attributable to their higher ploidy level. Independently-derived callus clones differed in their relative resistance to colonization by the fungus, and this was correlated with levels of medicarpin production. Two low molecular weight toxic fractions in the culture filtrate elicited medicarpin production in callus, stem tissue, seeds, and seedlings. Seedlings selected for their ability to germinate and grow in toxic culture filtrate were more resistant when inoculated with the live pathogen than were the non-selected parent plants (Latunde-Dada and Lucas, 1983). Alfalfa resistance to verticillium wilt 359 They, therefore, concluded that lowered sensitivity to wilt toxins is a useful component in selecting for field resistance. (iii) Indoor screening method developed at Lethbridge Research Centre (LRC) Huang and Hanna (1991) developed an efficient growth-room technique to evaluate alfalfa cultivars for resistance to verticillium wilt caused by V. albo-atrum. This method allows rapid screening of resistant plants from open pollinated alfalfa populations and so is used routinely for the development of resistant cultivars at LRC. For this purpose, germinated seeds of alfalfa are planted into Cornell Peat-Lite Mixes (Boodley and Sheldrake, 1977) in the Ferdinand style of Root Trainer BooksTM (Spencer-Lemaire Industries Ltd., Edmonton, AB). There are six cavities in each book and 16 books can be fitted into each standard tray (22 X 37 cm). Seedlings are grown in a growth room at 20 oC under a 16-h photoperiod and at 15 oC in the dark. Light intensity is maintained at 112-140 E s-1 m-2, using a mix of incandescent and fluorescent lamps. Plants are watered daily for 8-12 wk and then artificially inoculated. Conidia of an alfalfa strain of V. albo-atrum (LRS 112) collected from 10- to 12-day old cultures grown at room temperature on V-8 juice agar in Roux flasks are used for inoculation. A conidial suspension is prepared in sterile water containing about 7 x 106 to 10 x 107 spores mL-1. Prior to inoculation, plant shoots are trimmed to 4-5 cm in height. The books containing the plants are then removed from the trays and opened to expose the roots of all 6 plants. The roots of all plants are cut with a pair of dissecting scissors about 1.5 cm from the bottom of the book, without removing the plant from the book. Sixteen such books (96 plants) with injured plants are then placed in an upright position in an enamel tray (24 X 40 cm) containing 2L of the spore suspension. After the roots are soaked in the suspension for 10 minutes all the 16 books are replaced in a tray as before and kept in the growth room. For cultivar comparison, seedlings from each cultivar are normally replicated at least four times in a test when each replicate consists of at least 12 plants put in a pair of adjacent books. Approximately 4 weeks after inoculation, the plants are rated for symptoms of verticillium wilt, using a disease severity index (DSI). DSI is calculated based on a scale of 1 to 5 (Peaden, 1974), where 1 = plants with no symptoms and 5 = plants dead (Fig. 4). Plants rated 1 (no symptoms) and 2 (slight mottling on one or two leaflets) are classified as resistant (Peaden, 1974) and are normally transplanted into bigger pots and kept in the greenhouse or growth room for further observations. This method of screening has the following advantages over the growth room techniques described by others (Peaden, 1974; Graham et al., 1977; Busch and Smith, 1981; Busch et al., 1985; Elango et al., 1986): (1) Conserves growth room space and consequently energy requirement is low. About 1000 seedlings can be screened m-2 area compared to about 100 m-2 in other methods; (2) Reduces labour input since the plants are grown indoor and do not have to be dug up from field as is required in other methods; (3) Easy to do and survivors can be used for further observation or screened for resistance to other diseases such as bacterial wilt necessary for cultivar improvement; (4) Useful for breeding program as the survivors can be grown to adult stage and intercrossed for progeny testing and study of combining ability and (5) Resistant plants can be easily distinguished from susceptible plants with excellent repeatability (Fig. 5). 360 Surya N. Acharya & Hung-Chang Huang Figure 4. Severity of verticillium wilt in alfalfa (scale 1 to 5, left to right): 1= plants with no symptoms; 2= plants with slight mottling on one or two leaflets; 3= plants slightly stunted with more than two leaves showing yellowing; 4= sever stunting and leaf yellowing and 5= plants dead. Figure 5. Alfalfa cultivars showing differential resistance to verticillium wilt. Resistant cultivar (left), moderately resistant (centre) and susceptible cultivar (right) as per LRC developed indoor screening technique for verticillium wilt resistance. Using this screening method, many North American registered cultivars and experimental populations of alfalfa were tested for verticillium wilt resistance. Interestingly, the entries of earlier tests showed more susceptibility to the verticillium wilt pathogen compared to more recent tests where newer cultivars were included (Table 1 - 4). This is a remarkable achievement for the North American alfalfa breeders. Although not many new cultivars from European breeding programs were tested here, it would be safe to assume the availability of verticillium wilt resistant germplasm and emphasis on breeding for verticillium wilt resistance have improved resistance for this disease in European cultivars. (iv) Association of indoor method with field level resistance A study of resistance, using 2-wk-old seedlings root-dipped in a heavy spore suspension (5 x 106 spores/mL) from each of six alfalfa isolates of V. albo-atrum in separate tests, confirmed that Maris Kabul and Vertus showed significantly higher numbers of resistant plants than the susceptible DuPuits three weeks after inoculation (Huang and Hanna, 1991). Both Maris Kabul and Vertus were less resistant at 17-30 oC than at 12-21 oC (Heale, 1985). In each cultivar the level of seedling resistance was similar to the field resistance, although the correlation just failed to reach the level of significance using data for a single isolate. The extent of upward colonization of Vertus, Maris Kabul and the susceptible DuPuits, 21 days after inoculation, was estimated by Alfalfa resistance to verticillium wilt 361 incubating root and shoot pieces on agar. There was a marked limitation of colonization in Maris Kabul, which gave low wilt scores and a degree of tolerance with partial colonization in Vertus (Heale, 1985). The different resistance mechanisms detected here are probably accounted for by the recurrent field selections practised in the production of Vertus; selection was for apparently healthy plants (not necessarily uninfected); whereas, one of the parents, M. hemicycla used in the breeding of Maris Kabul appears to have contributed a defence mechanism which significantly limits host colonization (Heale, 1985). Percentage colonization for each of the cultivars was correlated with mean disease score. Non-pathogenic isolates from potato and tomato colonized seedlings of both cultivars after root-dipping; cut-stem-inoculated adult plants of all cultivars were also colonized to a significant degree, but without inducing symptoms (Heale, 1985). Table 1. Verticillium wilt resistance as determined by LRC developed indoor test on some popular cultivars in 1977. a DSI was based on 1=plants with no symptoms, 2=slight mottling on one or two leaflets. and 5=dead plants (Fig. 4); b%Plants rated as 1 and 2. Table 2. Verticillium wilt resistance observed on alfalfa entries included in 1985 western Canada Uniform Alfalfa Tests. a and b see footnote of Table 1. 362 Surya N. Acharya & Hung-Chang Huang Table 3. Verticillium wilt resistance observed on alfalfa entries included in 1999 western Canada WFTests. a and b see footnote of Table 1. Breeding methods used for development of verticillium wilt resistant alfalfa (i) Recurrent selection within commercial material: Panton (1965; 1967b) in Sweden showed that selection from Alfa, Tuna and Grimm, followed by hybridization, increased the frequency of resistant genes in the population. Moller and Andreasen (1975) used a similar approach in Denmark for developing resistance to this disease. Using Flemish alfalfa, Carr (1972) in Wales, UK developed the resistant cv. Sabilt. Selection for resistant plants within a winterhardy Flemish type Alfa and intercrossing the selected plants resulted in the development of the famous alfalfa cultivar Vertus in Sweden (Lundin and Jonsson, 1975). This cultivar has resistance to verticillium wilt and stem eelworm (Ditylenchus dipsaci). The resistant cultivar Lutece was selected from commercial material at Lusignan, France (Gondran, 1978). The cultivar Europe, widely grown in France and elsewhere in Europe, was selected for high yield from a Flemish type population, and had more wilt resistance than DuPuits (Heale, 1985). (ii) Hybridization between wild alfalfa and commercial lines: Alfalfa resistance to verticillium wilt 363 The diploid M. hemicycla was initially selected as highly resistant to verticillium wilt by screening a worldwide collection of wild alfalfa (Isaac, 1959; Heale, 1962). Chromosome doubled M. hemicycla plants were crossed with commercially grown M. sativa lines (DuPuits, Lahontan, Saranac and Flemish type). The resulting plants were then back-crossed repeatedly to M. sativa while selecting for resistance to the disease. This finally resulted in the highly resistant cultivar Maris Kabul which was released in 1970 (Heale, 1985). However, its yielding ability, resistance to other important diseases and winterhardiness are not comparable to other high yielding but verticillium wilt susceptible cultivars commercially grown in North America (Busch and Smith, 1981). Table 4. Verticillium wilt resistance observed in 2001 on alfalfa cultivars recommended for commercial production in Alberta. a and b see footnote of Table 1. Of these two approaches to the breeding of wilt resistant cultivars, the European experience would suggest that progress in North America towards a commercially acceptable, winterhardy cultivar with improved field resistance to verticillium wilt, is likely to occur more rapidly through selection within established home-bred material. Busch and Smith (1981) identified moderate levels of resistance which could form the basis for selection in hardy Canadian alfalfas. Elgin (1984) suggested that breeding alfalfa cultivars with 35 to 50% resistant plants will not be difficult. However, breeding an alfalfa cultivar with over 50% resistant plants was considered difficult (Miller and Christie, 1991). 364 Surya N. Acharya & Hung-Chang Huang Development of verticillium wilt resistant cultivars at LRC and its economic impact in western Canada Cultivar development At the Agriculture and Agri-Food Canada Research Centre, Lethbridge (LRC) development of verticillium wilt resistant cultivars combines indoor screening and field evaluation of the plants selected to be used as parents of synthetics. The process includes: 1) indoor screening for verticillium wilt symptomless plants from cultivars of diverse genetic base; 2) intercrossing of selected plants; 3) rescreening for symptomless plants and transplanting them to a field nursery infested with verticillium wilt inoculum; 4) selection for winterhardiness, yield and disease free plants in the field nursery; and 5) vegetative propagation of selected plants and intercrossing of the clones of selected plants. The following is a description of the development of one highly resistant and adapted alfalfa cultivar. AC Longview is one of the three high yielding verticillium wilt resistant cultivars developed at LRC (Acharya and Huang, 2000). The parental clones were derived from crosses between verticillium wilt resistant plants from nine North American bacterial wilt resistant cultivars (5444, A872, Anchor, Arrow, Beaver, Edge, Endure, Excalibur and Rambler) and three cultivars with high levels of resistance to both bacterial wilt and verticillium wilt (Barrier, AC Blue J and an experimental population LRC95CR-1). In 1989-90, plants were screened in growth rooms for resistance to verticillium wilt using the method of Huang and Hanna (1991) followed by a screening for bacterial wilt resistant plants using the method of Frosheiser and Barnes (1984). In 1990 winter, 135 selected plants were intercrossed by hand in the greenhouse. About 5000 progenies belonging to the 12 maternal cultivars were grown in a growth cabinet and reinoculated with verticillium wilt organism and scored for resistance. In 1991, approximately 2200 plants rated as free of verticillium wilt symptoms were transplanted to a disease nursery in Lethbridge, Alberta. After two years of observation and rouging, 865 vigorous and disease-free plants representing all 135 original selections were identified. Multiple clones of all entries were planted at random to facilitate inter-crossing. High density of leafcutter bees (M. rotundata) and hand pollinations were used to ensure a high level of inter-crossing among the selected plants and good seed set. This synthetic population (Syn 2) was then tested in field trials across western Canada for forage yield performance and released for commercial production. Verticillium wilt severity index of LRC developed alfalfa cultivars along with some cultivars recommended for western Canada are presented in Tables 3 and 4. Comparison of resistant and susceptible alfalfa cultivars for yield and quality In an experiment conducted in an irrigated commercial field naturally infested with V. albo-atrum near Lethbridge, Alberta, 12 alfalfa cultivars were seeded on July 9, 1986 using a four times replicated Randomized Complete Block Design (Huang et al., 1994). Ten of the cultivars were known to have some resistance to verticillium wilt and two, Beaver and Pacer, were known to be susceptible. Alfalfa plots were harvested as forage for 7 yrs (1987-1993) with two cuts in each year except for 1988, when three cuts were taken. The plots were harvested with a forage harvester and forage yield of each plot was expressed in dry weight basis. In 1993 (the 7th production year), the relative weed and alfalfa biomass were estimated before the first cut in all plots. For the purpose, two Alfalfa resistance to verticillium wilt 2 365 representative 0.25 m areas were hand-harvested and separated into alfalfa and weed components. Dry weights of the components were used to determine the composition of the samples. Prior to each cut in 1987, 1988 and 1989, stems of alfalfa plants with wilt symptoms were collected from each plot and stem segments were plated on selective medium (Christen, 1982) to verify infection by V. albo-atrum and to determine the incidence of diseased plants per plot. During these years, a colored marker was placed near the base of each infected plant to avoid repeat counting of diseased plants. In 1990 and during 1991-1993, the number of diseased plants in each plot was estimated by visual symptoms. The total number of plants in each plot was determined in 1990 and 1993. Data on number of diseased plants, percentage of stand diseased, plant stand, yield and weed data for each year were subjected to analyses of variance. The cultivars were clustered for similarity of their mean response within years, using a method described in Scott and Knott (1974). For the yield and disease response data, cluster analyses were done to reveal subsets of the cultivars that had similar mean disease levels and yield responses over the years (method 1 of Lin and Butler, 1990; Lin et al., 1992). On the basis of these results, the cultivars were grouped by disease levels. The incidence of verticillium wilt of alfalfa varied from year to year, and averaged over the years, the resistant cultivars Barrier, AC Blue J (tested as VW 34-2), 5444 and Vertus had a significantly lower incidence than the moderately resistant cultivars Maris Kabul, Admiral and Trumpetor and the susceptible cultivars Excalibur, WL316, Apollo II, Beaver and Pacer (Huang et al., 1994). Cultivars Excalibur, WL316 and Apollo II were considered resistant in other reports, but did not express resistance in this field test. Differences in yield were not significant until the third year. During the 7 yr trial, the total forage yields of resistant cultivars 5444, Barrier and AC Blue J were significantly higher than those of the susceptible cultivars Beaver and Pacer. The moderately resistant cultivars Maris Kabul, Admiral and Trumpetor had intermediate yields. Although the cultivar Vertus was resistant to the disease, its total forage yield was significantly lower than that of North American resistant cultivars 5444, Barrier, and AC Blue J. It was also clear that verticillium wilt can have significant impact on alfalfa production in southern Alberta, improvement in field level resistance can be achieved using the indoor screening method and selection for both disease resistance and adaptation are essential for optimizing forage production in this crop. Economics of growing verticillium wilt resistant cultivars Using the data from the above field study, Smith et al. (1995) calculated the yearly benefit of high resistance to verticillium wilt over moderately high resistance to be $21 ha-1 and over low resistance was $44 ha-1. In this case, loss due to reduced production and cost due to reduced length of stand were taken into account; while the Wyoming study only considered the loss in productivity over three years. The potential yearly benefits to producers in western Canada from the development and adoption of high verticillium wilt resistant cultivars were estimated at $2.2 million. The local benefits of growing adapted cultivars on irrigated land were $22 ha-1 yr-1. The regional benefits to western Canada from using adapted cultivars were estimated at $26.6 million yr-1. The greater potential benefit from growing adapted cultivars was attributed to benefits applied to all areas of western Canada; whereas, benefits from resistance to verticillium wilt were applied to areas known to be infested with the disease. 366 Surya N. Acharya & Hung-Chang Huang Conclusion Breeding for resistance to verticillium wilt in alfalfa, considered as the most efficient way to control the disease, has been very successful world-wide. The breeding program at LRC has used three sources of resistance and a rapid selection method for combating the disease. The resulting cultivars have shown higher levels of resistance than each of the cultivars used as the source population. This may have occurred due to pyramiding of the genes coding for different mechanisms rendering resistance to this disease. However, from a commercial stand point, breeding for resistance alone is not enough to optimize forage yield. Selection for disease resistance combined with improved yield and adaptation to western Canada has given the cultivars such as Barrier, AC Blue J and AC Longview an edge over other disease resistant but unadapted cultivars in terms of their commercial productivity. These cultivars can be used directly or as resistant germplasm in alfalfa improvement programs to reduce the impact of this devastating disease in alfalfa growing areas of the world. Acknowledgement The authors sincerely thank Dr. B. R. Christie, Retired Forage Breeder, Agriculture and Agri-Food Canada, Research Centre, Charlottetown, Prince Edward Island and Dr. R. Michaud, Agriculture and Agri-Food Canada Research Centre, Saint-Foy, Quebec, for their review of the manuscript and useful suggestions. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Acharya, S. N., and Huang, H. C. 2000. AC Longview alfalfa. Can. J. Plant Sci. 80:613-615. Arcieni, S., Pezzotti, M., and Damiani, F. 1987. In vitro selection of alfalfa plants resistant to Fusarium oxysporum f.sp. medicaginis. Theo. Appl. Gen. 74:700-705. Arny, D., and Grau, C. 1985. Importance of verticillium wilt of alfalfa in North America. Can. J. Plant Pathol. 7:187-190. Atkinson, T. G. 1981. 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