soybean rust provisional chapter utpal dey and g.p. jagtap

Provisional chapter

Soybean Rust

Utpal Dey and G.P. Jagtap

Additional information is available at the end of the chapter

1. Introduction

Soybean rust was first reported from Japan in 1903 (Sinclair and Shurtleff, 1975). It can causeyield loss up to 90 per cent (Anon., 1991). In India, the disease was first noticed at Pantnagarof Uttaranchal during 1970 and subsequently it was observed at Kalyani in West Bengal andthe foot hills of Uttar Pradesh. The disease was severe in 1970, 1971 and 1974 and mild in1972 and 1973 (Singh and Thapliyal.1977). It was disappeared then onwards and appearedsuddenly in epiphytotic form and caused substantial yield losses up to 80 per cent in North‐ern Karnataka and parts of Maharashtra during Kharif season 1994 and 1995 (Anonymous,1995 and Patil and Basavaraja, 1997). The disease appeared suddenly in epiphytotic form inrecent years (Kharif 1994 and 1995) and caused substantial yield losses particularly in partsof Karnataka, Maharashtra and Madhya Pradesh (Anahosur et al., 1995). Now, it has becomea major constraint for soybean production system particularly in northern Karnataka andparts of Maharashtra.

Soybean, a predominantly vegetarian society, such as India, fats and proteins of vegetableorigin are of special significance. The importance of soybean production in Indian agricul‐ture therefore is obvious. Soybean has been grown for centuries in the low hills of the Ku‐maun and Garhawal regions of the Himalayas, foot hills of Utter Pradesh and somescattered pockets in central India. However, the varieties grown have generally been of longmaturity duration, viny growth habit, with small and freely shattering pods, black seedsand very low yields. Seeds from these varieties were used primarily as pulses by the localpopulation and the green and dried vegetative parts were used as forage for cattle (Saxena,1976). In the world, soybean is cultivated over an area of 73.39 million ha with a productionof 161.90 metric tones and the productivity is 2206 kg/ha (Anon., 2004). The top five coun‐tries in the world are USA, Brazil, Argentina, China and India with respect to area and pro‐duction. In India it occupies an area of 8.87 m ha with the production of 9.46 m t andproductivity of 1069 kg/ha which is too low when compared to world productivity (Anony‐

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mous, 2007). In India major soybean producing states are Madhya Pradesh, Maharashtra,Rajasthan, Karnataka and Andhra Pradesh.

Several factors account for low productivity among them climatic conditions, differencesin rainfall pattern, outbreak of diseases and pests are important. Diseases play a majorrole in yield reduction. About 100 plus pathogens are known to affect soybean crop; ofwhich sixty six fungi, six bacteria, eight viruses and seven nematodes are involved (Sin‐clair, 1978). The world loss of more than seven million tonnes of soybean is due to diseas‐es alone (Sinclair, 1988). Among the diseases, rust (Phakopsora pachyrhizi Syd.), purple seedstain [Cercospora kikuchii. (T.matsu and Tomoyasu) Chupp.], anthracnose [Colletotrichumtruncatum (Schw.) Andrus and Moore], Myrothecium leaf spot (Myrothecium roridum Todeex Fries.), Rhizoctonia aerial blight (Rhizoctonia solani Kuhn), Alternaria leaf spot [Alternariatenuissima (Kunze ex Pers.)Wiltshire], powdery mildew (Microsphaera diffusa. Ke and Pk),collar rot (Sclerotium rolfsii. Sacc), bacterial pustule [Xanthomonas axonopodis pv. glycines(Nakano) Dye], and bud blight of soybean [Groundnut Bud Necrosis Virus (GBNV)] areconsidered the major constraint in production of soybean in India. These diseases causesconsiderable yield losses. 10-90% losses by rust (Bromfield,1984; Sinclair and Backman,1989), 15-30% by purple seed stain [Sinclair and Backman,1989), 16- 100% by anthracnose(Backman et al., 1982; Sinclair and Backman,1989), 35% by Rhizoctonia aerial blight (Sin‐clair and Backman,1989),10-35% by powdery mildew (Sinclair and Backman,1989), 30-40%by collar rot (Sinclair and Backman, 1989; Srivastava and Agarwal,1989), 11% by bacterialpustule (Patel and Kulkarni,1954; Sinclair and Backman,1989), and 25-100% by bud blight(Sinclair and Backman,1989;Srivastava and Agarwal,1989). Even though many options arethere for the management of these diseases such as cultural, chemical and biological meth‐ods; host plant resistance is the best, because of its eco-friendly nature and cost effective‐ness. In the host plant resistance, multiple disease resistance is more important anddesirable too, as they reduce losses caused by more than one disease. Identification ofmultiple disease resistant sources is also important as they can be utilized in breeding formultiple disease resistance.

2. Economic importance

Maximum yield losses in soybean due to soybean rust have been reported to range from10 to 90 per cent. It was predicted that annual yield losses for North America would be atleast 10 percent in the upper Midwest, Northeast, and Canada, and 50 per cent or greaterin the Mississippi Delta and the Southeastern states. It was also suggested that losses inheavily-infected areas anywhere in North America could exceed 80 per cent if effectivemanagement strategies were not used. It was observed in Madhya Pradesh in 1994 andcontinued to be a major threat in soybean cultivation until 1999, resulting in 80% loss inyield in susceptible varieties (Shanna and Mehta 1996), and it occurred in uredinial stagein Madhya Pradesh.

Soybean – A Review / Book 12

3. Symptoms

The rust is characterized by pustules, which start as numerous yellow lesions followed bythe appearance of a brown speck almost in the centre of these yellow lesions that soon de‐velop into light brown to dark pustules (Figure 1 & 2). Usually, on the lower leaf surface, onstems, several light brown elongated pustules appear, which upon rupture liberate massesof uredospores. Pustules may coalesce and turn dark brown to black in color. Very smalldark brown pustules are also produced on petioles and pods. As the disease advances yel‐lowing, pre-mature drying and defoliation is seen (Singh and Thapliyal, 1977).

4. Biology of the pathogen

There are approximately 80 species of Phakopsora known worldwide, six of which occur onlegumes. The latter species (P. meibomiae), commonly known as the cause of Latin Americanrust or Legume rust, is found in the western hemisphere and is not known to cause severeyield losses.

5. Distribution

Before 1992, soybean rust was known to cause significant losses in Asia and Australasia, in‐clusive of the following countries: Australia, India, Indonesia, Japan, Korea, Peoples Repub‐lic of China, Philippines, Taiwan, Thailand, Vietnam. Not much was documented about thedistribution of soybean rust in Africa before 1996 (given the problems with nomenclature);however, the following sequence of first reports were confirmed: Uganda, Kenya and Rwan‐da, 1996; Zimbabwe and Zambia, 1998; Nigeria, 1999; Mozambique, 2000; South Africa,2001. During 2001 P. pachyrhizi was detected in Paraguay (Morel et al., 2004) and this wasfollowed shortly by confirmation of its presence in Argentina in 2002 (Rossi, 2004) and Bra‐zil and Bolivia in 2003 (Yorinori, 2004). Uruguay, also a significant soybean producing coun‐try, recorded soybean rust for the first time in 2004 (Stewart et al., 2005). Soybean rust wasdetected in Hawaii in 1994 (Killgore and Heu, 1994) which stimulated the convening of aworkshop to discuss the potential threat that this held for the soybean crop in the U.S.A. Ascorrectly predicted by the delegates of this workshop (Sinclair et al., 1996), soybean rust hadthe potential to threaten crops on mainland U.S.A. In 2004, nine years later, Schneider et al.(2005) confirmed the presence of soybean rust in the U.S.A. From detection in Louisiana in2004, it spread to nine states by 2005, and was detected in 15 states in 2006 (Hartman, 2007).

The first report of the disease was from Japan in 1902. By 1934 the pathogen had been foundin several other Asian countries and as far south as Australia (Bromfield, 1980). In India,soybean rust was first reported on soybean in 1951 (Sharma, 1996). There have been severalearly reports of soybean rust in equatorial Africa (Bromfield, 1980), but the first confirmedreport of P. pachyrhizi on the African continent was in 1996 from Kenya, Rwanda, and Ugan‐

Soybean Rust 3

da. Since then, the pathogen has spread south with reports from Zambia and Zimbabwe in1998, Mozambique in 2000 and South Africa in 2001 (Levy et al., 2002). The westward move‐ment of the pathogen on the African continent was reported from Nigeria in 1999 (Akinsan‐mi et al, 2001). The first detection of P. pachyrhizi in the new world was in Paraguay inFebruary of 2001. The disease was found on soybeans in a limited number of fields in theParana River basin bordering Brazil. By 2002, soybean rust was widespread throughout Par‐aguay and in limited areas of Brazil bordering Paraguay, with reports of severe disease insome fields in both countries (Morel and Yorinori, 2002). The pathogen also was found in alimited area in northern Argentina (Rossi, 2003). During the 2003 growing season, the patho‐gen was detected in most of the soybean growing regions of Brazil with a conservative yieldloss estimate of 2.2 MMT, or approximately 5% of the annual production. In Paraguay, yieldloses from rust were limited due to dry conditions, while in Argentina the disease did notspread to the major production areas. The disease was found in Hawaii in 1994 on cultivatedsoybeans on the islands of Oahu, Kakaha, Kauai, and Hilo (1994).

6. Alternative hosts

The soybean rust pathogen is known to naturally infect 95 species from 42 genera of le‐gumes, inclusive of important weed species like Kudzu vine (Pueraria lobata) and major cropspecies such as common bean (Phaseolus vulgaris) (Wang and Hartman, 1992). Such a broadhost range is unusual amongst rust pathogens which normally have a narrow host range.The significance of the numerous alternative host possibilities for the soybean rust pathogenis that these may serve as an inoculum reservoir or a ‘green bridge’ from one soybean plant‐ing season to the next.

7. Epidemiology of soybean rust

The presence of a susceptible host, viable pathogen spores and suitable environmentalconditions are requisites for the development of a soybean rust epidemic. The optimumtemperature for urediniospore germination ranges between 12 and 27°C. Urediniosporegermination is greater in darkness, with light either inhibiting or delaying germination(Marchetti et al., 1976). A further requirement for urediniospore germination is a period ofleaf wetness. This period is considered to be about 6 h when this occurs within the opti‐mal temperature range. The optimum temperature for uredinia formation is reported byKochman (1979) to be 17°C (night) or 27°C (day). Uredinia form on the leaves nine dayspost infection (DPI) under these conditions, with the urediniospores maturing two tothree days later (Shanmugasundaram, 1999). The temperature in first week of August pri‐or to disease was 21.7°- 25.8°C, mean relative humidity 85.2%, rainfall 182 mm,4 rainydays in 7 cloudy days and during the first week of September, minimum temperature was21.8°-22.4°C, maximum 25.2°- 28.2°C, relative humidity 79-87% and rainfall 271 mm werethe favourable weather condition for the development of rust. This indicated that the high


rainfall and moderate temperature (22°-25°C) were significant when compared with oth‐ers. The rapid development of epidemic involving more number of repeated cycles of ure‐dospores indicating short latent period of the pathogen. Physiological age (pod formation)of the soybean plant plays a significant role in rust development (Bromfield 1984). How‐ever, severe infection of rust on volunteer soybean plants was observed at 4-leaf stage inChhindwara district in December. Dadke et al. (1997) reported infection of soybean plantsby rust at 15,30,45 and 60 days.

8. Disease cycle

In the presence of dew, each spore on a leaf can produce a germ tube, a slender thread thatgrows over the leaf surface. After about six hours, the germ tube penetrates the stomata andthe fungus begins to grow internally. A dew period of about six hours is sufficient for infec‐tion; longer dew periods are even more favorable. Rust develops best when temperaturesare 59-86° F.

Once the germ tube penetrates a leaf, water on the leaf surface is no longer essential and thefungus grows inside the leaf as a parasite, drawing water and nutrients from its host. Aboutseven days after infection, the fungus begins to produce spores just beneath the epidermison the lower side of the leaf. As more spores are produced, they burst through the epider‐mis. This is why the rust lesion is called a pustule. Spores that have ruptured the epidermisare exposed to the wind, which can transport them over long distances at high elevations.Spores do not seek out soybean or other host plants, but simply land by chance. Those land‐ing on susceptible hosts can cause new infections.

Airborne spores settle out of the air during still conditions, or are scrubbed out of the air byrain. The chance that a spore will be carried for long distances, land on a susceptible plant,and still be viable may be low. However, because a single acre of rusted soybean can pro‐duce more than 400 billion spores per day, the number of spores in the air over a healthysoybean field can be substantial, and an unlikely event becomes a near certainty. Phakopsorapachyrhizi cannot survive in crop residue. Spores may survive on their own for about 40days, but it is unlikely that spores on residue would survive an Indiana winter. Like otherrust fungi, soybean rust requires a living host to grow and reproduce.

9. Management practices

9.1. Cultural management

Cultural practices such as wider row width and reduced plant populations could poten‐tially decrease the severity of rust by decreasing the length of time leaves remain wet.Wider row width may also be beneficial for sprayer movement in the field and better cov‐erage of leaves throughout the crop canopy. It is not known how much of an impact these

Soybean Rust 5

methods may have on soybean rust. Growers should consider the impact of all diseasesbefore changing cultural practices. Continue to strive for high yields using proven bestmanagement practices for your fields, including selecting varieties to combat diseaseswhich are most yield limiting.

Highly resistant to rust SJ-1, resistant genotypes EC 241778, EC 241780, JS-19, RPSP-728,PK-838, PI-200492, PI-230970, Ankur, PI-462312, PI-459025, PI-230971 and PI-459024EC-241760, EC-333917, EC-325115, EC-251378, EC-389149 and EC-432536 moderately resist‐ant and susceptible genotypes DSb 1, Taifa Kaohsiung No.5 (TK-5) and JS 335. (Ramteke etal. 2004 and Verma et al. 2004, Patil et al. (2004)).

Hartman et al. (1991) inoculated P.pachyrhizi on two soybean genotypes at three different re‐productive growth stages (GS) in four trials. Rust was more severe on Taifa Kaohsiung No.5(TK-5), a commercial cultivar, than on SRE-B15-A (B15A). At GS R6, the rust infection wasranged between 14 to 95 per cent on TK-5 and 0 to 34 per cent on SREB15- A (B15A).

Hartman et al. (1992) evaluated 294 accessions representing 12 perennial Glycine spp. for re‐sistance to P.pachyrhizi and found that 23 per cent of these were resistant, 18 per cent weremoderately resistant and 58 per cent were susceptible.

Ramteke et al. (2004) conducted an experiment during the rainy season of 2002 and 2003 toscreen 41 genotypes of soybean [Glycine max (L.) Merrill] against rust under field conditionat rust hot spot Ugar-Khurd, Belgaum district, Karnataka and found none of the genotypesas resistant including seven differentials (PI-200492, PI-230970, Ankur, PI- 462312, PI-459025,PI-230971 and PI-459024) which were reported earlier as resistant.

Verma et al. (2004) evaluated 242 germplasm lines/cultivars of soybean under natural epi‐phytotic conditions for resistance to rust and reported only one line i.e., SJ-1 as highly resist‐ant, three lines viz., JS-19, RPSP-728, PK-838 as resistant, 16 lines as moderately resistant andrest were either susceptible or highly susceptible.

Patil et al. (2004) screened 1200 soybean genotypes to rust during kharif 2002 and 2003 and 36soybean genotypes against yellow mosaic during rabi/summer 1998-99 at Dharwad, San‐keshwar and Nippani regions of Karnataka. Out of 1200 genotypes screened against rust,only two genotypes EC-241778 and EC-241780 are reported as highly resistant and six geno‐types, EC-241760, EC-333917, EC-325115, EC-251378, EC-389149 and EC- 432536 moderatelyresistant and all the remaining genotypes as susceptible to highly susceptible. Among the 36genotypes screened against yellow mosaic, one genotype, DSb-4 was reported to be com‐pletely free from yellow mosaic. Early sowing (June end) of the crop received lesser damage(36.1 5%) when compared with crop sown in mid- and end of July.

9.2. Biological management

Plant derivatives possessing pesticidal properties are evoking worldwide interest as an al‐ternative or as supplements for the existing pesticides for several reasons (Toriyama, 1972).Integration of chemicals, plant extracts, and biotic agents along with resistance for managingplant diseases has considered as a novel approach (Papavizas, 1973). The literature on use of


plant extracts to manage soybean rust are lacking, so reviews pertinent to rust and other dis‐eases of different crops has been given

Maximum per cent inhibition of uredospore germination was observed with Azadirachta ind‐ica Juss. which was on par with Amaranthus viridis L. and Allium sativum L. at all the concen‐trations (2.5, 5.0 and 10.0%) tested and were significantly higher than the inhibition per centnoticed with other plant extracts. Wadhawani et al. (1986) reported complete inhibition ofuredospore germination of Puccinia helianthi in crude leaf extracts of Amaranthus spinosus,Lagenaria sicerari, Nerium indicum and Solanum nigrum. Antifungal properties of neem was al‐so established by Patil (2008).

Cristol 56SL and Cristol 47SL were biopesticides based on the botanicals from karanjia,neem and garlic fortified with the organic biodegradable emulsifiers (Personal communi‐cation from Krishna H. Gupta, Director, Krishna Antioxidants Pvt. Ltd.) and RBS 06 prod‐uct was chitin derivative from sea food with phenolic antioxidant extract (Personalcommunication from Dr. Kalidas Shetty, Professor of Food Biotechnology, Jefferson Sci‐ence Fellow at US State Department). Jones et al. (1989) reported that active principle forantifungal and antibacterial activity of Curcuma longa and A. indica assessed as the proteinpart of the plant extract.

Among the indigenous technology knowledge (ITK’s viz., panchagavya, vermiwash, cowurine, buttermilk and cow milk) are evaluated against uredospore germination of P. pachyr‐hizi at different concentrations the maximum inhibition of uredospore germination was ob‐served in cow urine followed by buttermilk and cow milk at 1:2 dilution. Whereas, cowurine and buttermilk treatments have shown on par relation with 1:5 and 1:10 dilutions. Un‐der field condition 1:10 dilution of cow urine was found effective in reducing the disease in‐tensity in different crops which has been well documented by several researchers (Sridhar etal., 2002; Manikandan, 2005; and Patil, 2007). Similarly buttermilk also found effective underfield condition and was also supported by many researchers (Arunkumar et al., 2002; Ribeiroet al., 2001; Zatarim, et al., 2005 and Patil, 2007). Patil (2007) reported three sprays of eithercow urine (1:10), cow milk (1:10), vermiwash (1:2) and panchagavya (3%) at 10 days intervalstarting from the onset of disease were found best among the different ITK’s tested in reduc‐ing rust severity and increasing the grain yield in soybean.

9.3. Chemical management

Three triazole fungicides have been found effective in the control of rust of soybean (Pa‐til and Anahosur, 1998). Dadke (1996) reported that hexaconazole (0.05%) was effectivein controlling the rust of soybean followed by triadimefon (0.1%), propiconazole (0.1%),difenconazole (0.1%) and mancozeb (0.25%). In the present study along with hexacona‐zole (0.1%), cristol 56 SL (1%), RBS 06 (1%) and cow milk (10%) have also reduced thedisease severity. Though the two sprays of hexaconazole fungicide alone has considera‐bly reduced the disease severity to higher level, but the addition of cristol 56 SL andcow milk in the spraying schedule along with hexaconazole was found effective in re‐ducing the per cent disease index. In addition, integration of RBS 06 with hexaconazole

Soybean Rust 7

at Dharwad and neem oil with hexaconazole at Ugar-Khurd also proved better in mini‐mizing the per cent disease index. Anahosur et al. (2000) reported that use of nimbici‐dine (0.5%) inter mixed with hexaconazole (0.1%) sprays reduced the rust disease indexand thereby increase the yield of soybean. Hexaconazole, triadimefon and propiconazoleat 0.1 per cent sprayed at 15 days interval starting from the onset of disease were foundeffective in reducing soybean rust severity, with significant increase in seed yield and100 seed weight. Highest benefit cost ratio of 9.3 was recorded with hexaconazole fol‐lowed by propiconazole (4.0) and triadimefon (1.9) (Patil and Anahosur, 1998). nimbici‐dine in the spray schedule along with hexaconazole, propiconazole and triadimefon werefound effective in reducing the soybean rust severity with increasing seed yield, 100 seedweight and B: C ratio (Gurudatt et al. 2003). Two sprays of triadimefon (Bayleton @0.1%) were most effeetive, as these completely checked the rust infection and increasedthe yield (32%) over the control. Highest benefit : cost ratio (0.12) was observed undermancozeb (Indofil M-45,0.25%). Soil application of sulphur @ 20 kg/ha along with recom‐mended dose of N P K @ 20 kg/ha, P2O5 kg/ha 50 kg/ha and K2O 20 kgha) significantlyreduced disease index (%) but farmyard manure with N P K resulted in maximum seedyield of 1.364 and 1.376 tonneslha at Chhindwara and Seoni respectively).

10. Isozyme studies

Isozymes are the enzymes with the same catalytic activity but different molecular structure.The peroxidase enzyme is believed to be contributing to the resistance by the oxidation ofphenolic compounds to quinones, which are toxic to micro-organisms (Clark and Lorbeer,1975; Sempio et al., 1975b and Urs and Dunleavy, 1975). Generally, the peroxidase and poly‐phenol oxidase enzymes have a defensive role against the invading pathogen, in that it isresponsible for removal of toxic hydrogen peroxide in the host cells, there by protecting thecells from getting damaged.

11. Peroxidase

The results of the study are presented in the Table 1. The study revealed that at 45 daysafter sowing in both healthy and diseased leaf, in each genotype two bands appeared ex‐cept in JS 335 where no band appeared in healthy leaf sample but all the two bands ap‐peared under diseased conditions. However, the Rm values of first band (0.81) andsecond band (0.83) was same in all the genotypes. At 75 days after sowing, only oneband appeared under both healthy and diseased conditions. However both polymorphicisozyme bands were observed under diseased condition with Rm values 0.82, 0.84, 0.85and 0.86. Whereas in case of healthy leaf conditions monomorphism existed with Rmvalue of 0.83.


Figure 1. Small dark brown pustules produced on leaves, petioles and pods

12. Polyphenoloxidase

The study revealed that at 45 DAS under both healthy and diseased conditions in each geno‐type only one band appeared. At 45 DAS, under both healthy and diseased conditions Rmvalue remained same 0.87 (Table 2. At 75 DAS polymorphic bands were observed in healthyand diseased conditions. Under healthy condition, resistant genotypes EC 241778 and EC241780 recorded maximum Rm values of 0.89 and least Rm value of 0.86 was observed in JS335. Under diseased condition EC 241780 recorded maximum Rm value of 0.93 and least Rmvalue of 0.90 was recorded in DSb 1.

Genotypes

Peroxidase

45 DAS 75 DAS

H DH D

B1 B2 B1 B2

JS 335 - - 0.81 0.83 0.83 0.82

DSb 1 0.81 0.83 0.82 0.84 0.83 0.84

EC 241778 0.81 0.83 0.83 0.85 0.83 0.85

EC 241780 0.81 0.83 0.83 0.85 0.83 0.86

DAS: Days after sowing H: Healthy D: Diseased B: Band

Table 1. Relative mobility (Rm) values of peroxidase at different crop growth stages of soybean genotypes.

Soybean Rust 9

Genotypes

Peroxidase

45 DAS 75 DAS

H D H D

JS 335 0.87 0.87 0.86 0.91

DSb 1 0.87 0.87 0.87 0.90

EC 241778 0.87 0.87 0.89 0.92

EC 241780 0.87 0.87 0.89 0.93

DAS: Days after sowing H: Healthy D: Diseased

Table 2. Relative mobility (Rm) values of polyphenol oxidase at different crop growth stages of soybean genotypes

Pale yellowish-brown urediniospores, median view.

Red brown soybean leaf lesions with uredinia Soybean leaves with uredinia and some chlorotic areas

Cross-section of leaf showing telium with two-three layers of teliospores

Figure 2. Symptoms of soybean rust


1. JS 335 2. D Sb 1 3. EC 241778 4. EC 241780

Figure 3. RAPD pattern of primers on rust resistant and susceptible genotypes of soybean

Soybean Rust 11

13. Enzymatic activity in rust resistant and susceptible genotypes

a. Phenylalanine Ammonia Lyase (PAL) activity (µg of cinnamic acid mg-1 of protein hr-1)in leaf

The enzyme activity of Phenylalanine Ammonia Lyase (PAL) in two resistant and two sus‐ceptible genotypes at different hours after inoculation with uredospores of Phakopsora pa‐chyrhizi are presented in Table 3. The PAL activity showed much variations among foursoybean genotypes. At zero hour (immediately after inoculation), the highest PAL activitywas observed in susceptible genotype DSb 1 (9.13) followed by JS 335 (8.91). However, theminimum Phenylalanine ammonia lyase activity was observed in resistant genotype EC241778 (6.70) followed by 6.98 in case of EC 241780. Again the susceptible genotype DSb 1showed maximum PAL activity at II stage (6 hours after inoculation) followed by JS 335(8.73). At the same interval the least PAL activity was observed in resistant genotype, EC241778 (6.91) followed by EC 241780 (7.19).

At stage III (12 hours after inoculation), the maximum PAL activity was observed in JS 335(8.64) followed by 8.62 incase of DSb 1. The least PAL activity was observed in EC 241778 (7.14)followed by 7.41 in case of EC 241780 for the corresponding period of interval. The enzymePAL had its highest activity in resistant genotype EC 241780 (8.33) at stage IV (24 hours after in‐oculation), followed by DSb 1 (8.30), the least PAL activity was observed in EC 241778 (8.01). Atstage V (48 hrs after inoculation) again the genotype EC 241780 showed maximum PAL activi‐ty of 10.18 followed by EC 241778 (9.31). The least PAL activity was observed in susceptiblegenotype JS 335 (7.26) followed by DSb 1 (7.61). At stage VI (72 hr after inoculation), PAL en‐zyme had a maximum activity of 11.95 in case of resistant genotype EC 241780 followed by EC241778 (10.26). The least PAL activity was observed in case of susceptible genotype JS 335 (6.50)followed by DSb 1 (6.91). Overall, there was a decrease in PAL activity from stage-I to stage-VIin both the susceptible genotypes viz. JS 335 and DSb 1 (27.05 and 32.13%) respectively. Howev‐er, the PAL activity was increased from stage-I to stage-VI in both resistant genotypes viz. EC241778 and EC 241780 (34.69 and 41.59%) respectively.

b. Tyrosine Ammonia Lyase (TAL) activity (µg of p-coumaric acid mg-1 of protein hr-1) inleaf

The TAL activity (Table 4) was to its maximum in susceptible genotype DSb 1 (25.37) atstage I (immediately after inoculation) followed by 24.69 incase of JS 335. Whereas the activi‐ty was minimum in EC 241780 (24.04) followed by EC 241778 (24.15). At stage II, again max‐imum TAL enzyme activity was observed in susceptible genotype DSb 1 (25.67) followed byJS 335 (24.37). The least TAL enzyme activity was observed in resistant genotype EC 241778(24.33) followed by EC 241780 (24.34). The TAL enzyme had a maximum activity of 24.42 inDSb 1 followed by JS 335 and least activity was observed in EC 241780 (24.01) followed byEC 241778 (24.12) stage III, and same trend continuous at stage IV also. At stage V, TAL en‐zyme had a highest activity in resistant genotype EC 241778 (27.81) followed by EC 241780(27.72). The least TAL enzyme activity was observed in susceptible genotype JS 335 (20.46)followed by DSb 1 (21.92).


Again at stage VI the TAL enzyme had a maximum activity in case of resistant genotype EC241778 (29.90) followed by EC 241780 (29.81). The least TAL enzyme activity was observedin susceptible genotype JS 335 (18.97) followed by DSb 1 (20.47). Overall, there was a de‐crease in TAL activity from stage-I to stage-VI in both the susceptible genotypes viz. JS 335and DSb 1 (30.15 and 23.94%) respectively. However, the TAL activity was increased fromstage-I to stage-VI in both resistant genotypes viz. EC 241778 and EC 241780 (19.23 and19.26%) respectively.

Genotypes

PAL activity (µg cinnamic acid mg-1 protein hr-1)

Time interval (stages) after inoculation (hr)

I stage

(0 hr)

II stage

(6 hr)

III stage

(12 hr)

IV stage

(24 hr)

V stage

(48 hr)

VI stage

(72 hr)

Per cent

change

JS 335 8.91 8.73 8.64 8.12 7.26 6.50 -27.05

DSb 1 9.13 8.93 8.62 8.30 7.61 6.91 -32.13

EC241778 6.70 6.91 7.14 8.00 9.31 10.26 34.69

EC241780 6.98 7.19 7.41 8.33 10.18 11.95 41.59

Table 3. Specific activity of phenylalanine ammonia lyase (PAL) in soybean genotypes at different hours afterinoculation with P. pachyrhizi.

GenotypesTAL activity (µg cinnamic acid mg-1 protein hr-1)

Time interval (stages) after inoculation (hr)

I stage

(0hr)

II stage

(6 hr)

III stage

(12 hr)

IV stage

(24 hr)

V stage

(48 hr)

VI stage

(72 hr)

Per cent

change

JS 335 24.69 24.37 24.13 22.93 20.46 18.97 -30.15

DSb 1 25.37 25.67 24.42 23.17 21.92 20.47 -23.94

EC241778 24.15 24.33 24.12 25.87 27.81 29.90 19.23

EC241780 24.04 24.34 24.01 25.69 27.72 29.81 19.36

Table 4. Specific activity of tyrosine ammonia lyase (TAL) in soybean genotypes at different hours after inoculationwith P. pachyrhizi

Velazhahan and Krishnaveni (1994) observed higher activities of peroxidase and polyphenoloxidase in the resistant cultivar (No 179) of sunflower than the susceptible cultivar (EC68414) following infection with P. helianthi. In infected leaves of the susceptible cultivarthere was an increase in peroxidase activity but the ratio of peroxidase activity decreasedduring the later periods of infection.

Yurina et al. (1996) determined the peroxidase activity in the leaves of six wheat cultivarsdiffering in their resistance to powdery mildew Erysiphe graminis f. sp. tritici Marchel.

Soybean Rust 13

The pathogen stimulated the peroxidase activity in all six cultivars, but to a greater extent inthe resistant ones.

Leach et al. (1998) reported that in rice plants the infection due to Xanthomonas oryzae pv.oryzae Uyeda and Ishiyama increased the secretion of cell wall precursors and enzymes suchas peroxidase.

Kalappanavar and Hiremath (2000) recorded higher Rm values at all the stages of cropgrowth in multiple foliar disease resistant sorghum genotypes. Further he observed pro‐duction of two isozyme bands of both peroxidase and polyphenol oxidase in resistantgenotypes where as in susceptible genotypes only one isozyme band was produced at 80days after sowing.

Malli et al. (2000) observed that in mothbean the peroxidase activity decreased significantlyin susceptible genotypes than in resistant genotypes with the increasing intensity of mung‐bean yellow mosaic virus infection.

Chakraborty et al. (2002) studied the isozyme pattern of peroxidase, polyphenol oxidaseand indole acetic acid oxidase by polyacryl amide gel in tea plants induced by foliar in‐fection with Exobasidium vexans Masse and showed four isozymes in healthy and five ininfected leaves.

In disease resistance mechanism phenylalanine ammonia lyase (PAL) and tyrosine ammonialyase (TAL) play an important role in the conversion of phenylalanine and tyrosine to cou‐maric acids. These provide the phenyl propane carbon skeleton for the synthesis of flavo‐noids phenolic phenyl propanes and lignin. PAL, the key enzyme of phenolic biosynthesiswas first reported in Hordium vulgare L. by Koukol and Conn (1961). It catalyses the deami‐nation of phenylalanine and by antiperiplanar elimination of the Pro-S-Proton from carbon 3and of NH2 from carbon 2, it produces trans-cinnamic acid. The enzyme PAL has an aver‐age molecular weight of 3,30,000 at optimum pH of 8.8 and has no cofactor requirement.Some preparations of PAL especially those from grasses show activity towards tyrosine, butit has not yet been established whether there exists a separate enzyme for deamination oftyrosine.

In general, PAL is otherwise highly specific towards its named substrate. However undercritical conditions of rapid plant growth, phenylalanine will be preferentially incorporatedin to proteins before it becomes available for incorporation in to flavonoides and other phe‐nolics viz., PAL. Presumably, under conditions when protein synthesis is minimal more phe‐nylalanine becomes available for conversion into phenolics (Margha, 1977).

In general resistant varieties are characteristics of rapid conversion of phynylalanine and ty‐rosine to coumaric acids. In test with two susceptible and two resistant cultivars, high activi‐ty of phenylalanine ammonia lyase (PAL) was associated with the varieties resistant to redrot (Glomerell tucumanensis (Speg.) Von Arx and Muller). Conversely, low activity of theseenzymes was noticed in varieties susceptible to the disease. (Madan et al. 1991).

Boonchitsirikul et al. (1998) recorded the time course changes of enzyme activities of phenyl‐aianine ammonialyase (PAL) and tyrosine ammonia lyase (TAL) in young rice panicles ino‐


culated with the blast fungus Pyricularia grisea (Cooke) Sacc. were examined during a fiveday period after inoculation and compared with those of uninoculated controls. Four ricematerials (2 cultivars and 2 F8-lines selected for blast resistance and showing differences inthe resistance to blast disease) were used for the experiment. The activities of PAL and TAL1 or 2 days after inoculation increased in the inoculated as well as in the control panicies andreached maximum values within 2 or 3 days. The increase of the ratios and levels of the ac‐tivities of PAL and TAL in the inoculated samples were much higher than those of the unin‐oculated controls. TAL activity was considerably lower than the PAL activity and the timerequired for reaching the maximum activity in 7F8-8 and Reiho was delayed by 1 day com‐pared with the PAL activity. Both the ratios and levels of increased activity of PAL and TALwere significantly different among the four rice cultivars harbouring different blast resist‐ance genes.

Kale and Choudhary (2001) studied the expression of PAL activity in groundnut cultivarGirnar-1 (resistant to) and TAG-24, ICGS-10, ICGS-76 and SB-XI susceptible to early and lateleaf spot diseases. Resistant interaction was correlated with early and rapid inoculation ofPAL as evidenced from the enzyme activity. The PAL was expressed differentially in differ‐ent parts of seedling of all cultivars, however, the response of hypocotyles was maximum inresistant cultivar Girnar-1.

Gupta and Kaushik (2002) reported higher specific activities of PAL and TAL in diseasedleaves and siliqua walls compared to healthy leaves in all four varieties of mustard, andwere also higher in leaves compared to siliqua. The increased activity following infectionsuggests their possible involvement in providing resistance against disease.

Chakrabarty et al. (2002) concluded that induced rather than constitutive levels of PALplayed crucial role in governing resistance in cotton genotypes. The magnitude of inductionwas invariably higher in resistant lines than in the susceptible plants. Besides the ability ofresistant plants to prevent greater loss in PAL upon infection appeared important for resist‐ance.

Sharma (2003) reported high activity of phenylalanine ammonia lyase (PAL) and tyrosineammonia lyase (TAL) in highly resistant healthy (uninoculated) apple rootstock (MM 115)and minimum activity of PAL and TAL were recorded from highly susceptible ones(MM103, MM104). The activities of PAL and TAL enzymes increase rapidly in resistant root‐stocks (MM115, M25) at the initial stages of pathogenesis and subsequently declined rapid‐ly. The activity of these enzymes also continued to increase gradually with pathogenesis (upto the twentieth or twenty-fifth day of inoculation) in highly susceptible rootstocks. In resist‐ant rootstocks, the activities of enzymes remained higher during pathogenesis in compari‐son to that of susceptible ones.

Soma Das et al. (2003) studied the role of lignification and the enzymes involved in the bio‐synthesis of lignin i.e., phenylalanine ammonia lyase (PAL) in the wheat spot blotch diseaseresistance using a resistant (Pusa-T 3336) and a susceptible genotype (Agralocal). PAL activ‐ity began to increase in both the genotypes at 12 h after inoculation and reached to maxi‐mum at 2 days after inoculation. In resistant genotype Pusa-T 3336, 30-fold increase was

Soybean Rust 15

found at 12h after inoculation, whereas in susceptible genotype Agra local, the activity wasmarginal. High lignification was observed in the resistant genotype.

14. DNA fingerprinting of rust resistant and susceptible genotypes ofsoybean

Abundance and uniform distribution of genetic markers in any plant species is necessary fora number of applications like varietal fingerprinting, marker assisted selection and numberof other studies including diversity analysis at various levels. Presently DNA based markersare a class by themselves. Almost unlimited in number they are widely and evenly distribut‐ed in the genome. Unaffected by other genes and environment, the genotype of any individ‐ual of the population with respect to DNA based markers can be determined unequivocally,at any stage of the development non-destructively. In addition, it is possible to generatemarkers to suit specific applications without altering the genotype of the individuals. Theinformation about molecular polymorphism is reviewed here with special emphasis on ran‐domly amplified polymorphic DNA (RAPD).

Among the 60 arbitrary decamer primers screened 57 primers showed clear and scorableamplicons in each DNA sample with few ghost or minor bands, which were ignored. Sam‐ple gels resulting from arbitrary primers used across individually pooled genomic DNA ofall the four genotypes is presented in Figure 3. Among the 60 primers screened 55 primersshowed polymorphism, two primers produced monomorphism (OPA 16 and OPF-11) andthe remaining three primers did not show any amplicons. A total of 263 amplicons resultingfrom 57 primers were available for scoring analysis. Out of these, total of 144 polymorphicbands were observed. The primer OPF-18 produced maximum of 87.50 per cent polymor‐phism. The highest number of eight amplicons per primer were produced by three primersviz., OPA-03, OPF-13 and OPF-18 followed by seven amplicons by OPB-09, OPF-02 andOPF-17 primers. The lowest of one amplicon was amplified by OPA-16 and OPF-11 primer.On an average there were 4.38 amplicons per primer of which 2.40 were polymorphic, indi‐cating variability among four soybean genotypes.

Based on simple matching co-efficients, a genetic similarity matrix was constructed to assessthe genetic relatedness among selected soybean genotypes. Similarity co-efficients rangedfrom 0.70 to 0.86 among genotypes. The minimum genetic relatedness was 70 per cent be‐tween DSb 1 and EC 241778 genotypes. The maximum genetic similarity of 86 per cent wasevident between the genotypes of JS 335 and EC 241780. Genetic similarity coefficients indi‐cating the extent of relatedness among genotypes are furnished in Table 5.

Further, clustering analysis clearly showed two major groups X and Y in genotypes at a sim‐ilarity co-efficients of 0.74 (Fig. 4). The first group (X) comprised three genotypes while thedistinct second group (Y) consisted of only one genotype (EC 241778). The group X is subdi‐vided into X1and X2 at a similarity co-efficients of 0.80 where X1 at a similarity coefficientsof 0.80 comprised only one genotype (DSb 1). The X2 group comprising two genotypes (JS335 and EC 241780) at a similarity coefficient of 0.86.


15. Randomly Amplified Polymorphic DNA (RAPD)

The RAPD technology has quickly gained wide spread acceptance and application becauseit has provided a relatively simple tool for genetic analysis in biological systems. However,RAPD is the best assay when the nucleotide sequence is not known. Unlike other PCR pro‐tocols, which utilize two primers of defined sequence, RAPD detects nucleotide polymor‐phism using only one primer of an arbitrary nucleotide sequence. It has been successfullyused for cultivar analysis in a number of plant species.

Genotypes JS 335 DSb 1 EC 241778 EC 241780

JS 335 1.00 - - -

DSb 1 0.82 1.00 - -

EC 241778 0.79 0.70 1.00 -

EC 241780 0.86 0.79 0.74 1.00

Table 5. Similarity co-efficients of soybean genotypes obtained by random primer PCR analysis.

Figure 4. Dendrogram showing genetic relatedness among rust resistant and susceptible genotypes of soybean

The study of the results are presented in Table 6. The study revealed that, out of 60 primersscreened, only eight showed polymorphism which are consistent in rust resistant and sus‐ceptible genotypes. However in both susceptible genotypes (JS 335 and DSb 1) totally sixprimers viz., OPA-09, OPB-05, OPB-20, OPF-14, OPF-18 and OPF-20 showed polymorphicbands but absent in case of both the resistant genotypes (EC 241778 and EC 241780). Amongthese primers, OPF-20 produced maximum of two polymorphic bands whereas remainingfive primers produced only one polymorphic band. The maximum per cent polymorphismwas observed in OPA-09 and OPF-20 (33.30%) followed by OPF-20 (25%). However the leastpolymorphism per cent was observed in OPF-18 (12.50%).

Soybean Rust 17

Arbitrary

primers

Bands present in resistant

but absent in susceptible

genotypes

Bands present in

susceptible but absent in

resistant genotypes

Per cent

polymorphism per

primer

OPA-09 - 1 33.30

OPB-05 - 1 20.00

OPB-08 1 - 25.00

OPB-17 1 - 50.00

OPB-20 - 1 33.30

OPF-14 - 1 16.00

OPF-18 - 1 12.50

OPF-20 - 2 25.00

Table 6. RAPD primers differentiating rust resistant and susceptible genotypes

The study also revealed that, among 60 primers screened, only two primers (OPB-08 andOPB-17) shown polymorphic bands in both the resistant genotypes (EC-241778 and EC-241780). But absent in both the susceptible genotypes (JS 335 and DSb 1). However, both theprimers shown single polymorphic band. However the per cent polymorphism was maxi‐mum with primer OPB-17 (50.00%). The primer OPB-08 showed 25.00 per cent polymor‐phism was observed. Discrimination power of the primers The values of discriminationpower (%) calculated for eight primers which are consistent for differentiating rust resistantand susceptible genotypes. The highest differentiation power of 100 per cent was observedin the primers OPA-09, OPB-05 and OPB-08. These primers produced distinct RAPD band‐ing patterns for all the four soybean genotypes used in the study.

Quinshang et al. (1999) conducted preliminary studies on identifying RAPD marker associat‐ed with resistant gene to this disease. DNAs from resistant genotype AGS 129 other isogeniclines and susceptible genotype NS-1 were extracted and amplified by using 80 operon pri‐mers. Totally 365 bands, 67 of which showed polymorphism, were amplified. Two polymor‐phic bands designated OPA02710, OPH02800, respectively, was considered as potentialRAPD markers linked to resistance to soybean downy mildew. Because OPA0270 justshowed in resistant AGS 129 and other isogenic lines, OPH02 800 just in susceptible NS1genotype.

Rizi et al. (1999) were studied the level of genetic variation among two hexapliod wheat (Eta,D15) and two soybean cultivars (Williams, A3935) of divergent geographic origin were test‐ed using RAPD (Randomly Amplified Polymorphic DNA) assay. For wheat out of 80 arbi‐trary decamers screened, two produced polymorphic patterns, 56 were monomorphic forboth cultivars and 11 did not prime any amplification product. For soybean out of 40 arbi‐trary decamers screened, three produced polymorphic patterns, 33 were monomorphic forboth cultivars and two did not prime any amplification product. The level of genetic varia‐tion was calculated for both species and it is respectively 3.6 per cent for wheat and 7.9 percent for soybean cultivars.


Wang et al. (1999) conducted studies in identifying RAPD marker related to resistance Vssusceptibility to soybean cyst nematode (Heterodera glycines Ichinohe, Sen) Race 1 which isdominant in shandong province China. The 64 plants from five BC, F2 lines out of the 104ones were used for RAPD analysis. The total genomic DNA extracted according to theCTAB method. Equal amount of DNA to the CTAB method. Equal amount of DNA from allthe 12 resistant plants out of the 64 ones from five BC, F2 lines was mixed to make a resist‐ant bulk, and five susceptible bulks were formed in a same way corresponding to the fivelines. Total 320 primers (operon technologies, Inc. Almeda, CA) were screened for those pro‐ducing polymorphic bands. Among them OPAO19 was found to produce 1.2 kb polymor‐phic band designated as OPAO191200, and then was used to amplify yenomic DNA fromthe parents and each of the 64 BC, F2 plants. The OPO191200 hand could be produced in7605, al the susceptible plants of the BC, F2 line no. 11 and most susceptible plants of line 43,but not in all resistant plants. It was inferred that the BC, F, plants line no. 11 and 43 had asusceptible locus which is not existed in resistant lines.

Zhao et al. (2000) used RAPD technology to construct DNA fingerprints of 64 genotypes ofsoybean in Jilin province. Repeated amplification and selection of 140 primers resulted insuccessful construction of DNA fingerprints for all the genotypes studied. Primers OPA-04,OPG-07, OPO-11 and OPF-014 showed good polymorphism, each having 6-12 bands, with amolecular weight in the range of 0.2-3 kb. OPA-04 appeared the best primer being able todistinguish 40 of the genotypes.

Naik et al. (2002) were successfully applied DNA fingerprinting to distinguish two popularmulberry (Morus spp.) cultivars viz., Mysore local and V-1. RAPD analysis of 12 collectionsof each of these two cultivars derived from clones of different sources using 12 oligonucleo‐tide random primers generated 73 applicons of which 40 were monomorphic and the rest 33were polymorphic (45%). All the primers produced typical banding profiles for each of thecultivar suggesting the usefulness of the technique in DNA fingerprinting and cultivar iden‐tification. The genetic distance between these two cultivars based on the RAPD data set wasestimated as 0.292, which is low in comparison to the morpho-agronomical difference, sug‐gesting a narrow genetic base of the crop.

Author details

Utpal Dey and G.P. Jagtap

Marathwada Agricultural University, Parbhani, Maharashtra, India

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