Passalora sojina (leaf spot of soyabean)

Young plants of susceptible cultivars show the earliest symptoms on leaves, stems and pods. On artificially inoculated plants, the first symptoms of P. sojina are visible as early as 8 days after inoculation under greenhouse conditions, and after 9 days for field inoculations (Yorinori, 1989).Leaf SymptomsSymptoms of leaf spot may vary from one cultivar to another. The youngest fully developed leaves at the time of infection with P. sojina will show the greatest number of lesions. Leaf spots are larger on younger leaves and are smaller in size as the plant grows towards maturity. There is no progressive increase in lesion size once the lesions become visible. However, the colour of the lesions changes. When they are first visible, they appear as white-green, water-soaking or wilting spots, evolving to greyish-green, circular to subcircular, varying in size from minute spots to 5 mm in diameter (Yorinori, 1980). Individual lesions may coalesce to form large, irregular lesions.The difference in lesion colour between the lower and upper leaf surfaces is an important feature for diagnosing frogeye leaf spot in the field. On the upper leaf surface, as the lesions become older, necrotic tissue progressively turns reddish-brown, light-brown and finally paper-white. Fully developed lesions may present an outer thin yellow halo surrounding a reddish-brown margin and a reddish-brown to light-brown centre. On the lower leaf surface, the lesion colour varies from light-green to greyish-green and finally to light-brown, usually with a black dot in the centre, where conidiophores form during sporulation. The darker (greyish to brownish) colour of the lower leaf surface is due to sporulation, which may occur 24 hours after the lesions are first visible. Lesions formed on leaves in the shaded portion of the lower canopy may have sporulation on both sides.P. sojina causes pathological changes in the host by producing substances that act in advance of hyphal growth. The host cells first show a change in staining reaction followed by protoplasmic disorganization and complete collapse of the affected cells (Lehman, 1928). In infected leaf tissues, no mycelium could be detected among the affected cells during the early development of the lesions, where there was some collapse of tissue. Later, as necrosis developed at the centre of the lesions, the mycelium grew intercellularly and gave rise to conidiophores, from the stromata on both leaf surfaces. Soyabean leaf tissue infected by P. sojina shows cellular disorganization and accumulation of chlorophyll, starch and labelled phosphate in advance of the mycelium in a distinct plesionecrotic ring around the holonecrotic area constituting the frogeye leaf spot (Benedict and Fucikovsky, 1966).Severe foliar infection by P. sojina results in early defoliation, poor grain formation and uneven maturation. Stem SymptomsOn the stem, the lesions start as small <1 mm diameter) reddish-brown spots and enlarge to an elliptical shape more than 1 cm long. Older lesions have a thin dark reddish-brown margin and progressively lighter colour in the centre. When environmental conditions are favourable for sporulation, a dark-grey layer of conidiophores forms in the centre of the lesion. In stems, P. sojina is mainly confined to the cortex. Injury to the phloem and cambium is usually caused by diffusion of the toxic substances from the necrotic cortex (Lehman, 1934).Pod SymptomsIn pods, the mycelium penetrates the pod wall, entering the thin white membranes lining the pod. Growth of the fungus is usually superficial on the seeds (Lehman, 1934).The development of lesions on the pods is similar to that on the leaves. Symptoms are first visible as water-soaking and circular spots of various sizes, which progress to dark-grey spots. On the pods, the lesions vary in sizes from 1 to 5 mm in diameter, they are circular or slightly elongate and may develop light-brown discoloration in the centre. Under heavy infection, the lesions may coalesce and turn dark-grey, resulting in rotting of the pod and seeds (Sinclair, 1982; Yorinori, 1980).Seed SymptomsEarly defoliation results in smaller seeds with green seed coats. Further reduction in the seed quality is caused by other associated seedborne pathogens such as Cercospora kikuchii, Diaporthe phaseolorum var. sojae, Colletotrichum truncatum and Fusarium spp.Seeds infected by P. sojina develop conspicuous light to dark-grey or brown areas that vary from minute specks to large blotches covering the entire seed coat. Some lesions show alternating bands of light and dark brown. Infected seeds usually show cracking of the seed coat (Sinclair, 1982). Damage to the seed depends on the susceptibility of the cultivar and the growth stage when pod infection occurred. Leaf, stem and pod may differ in susceptibility to P. sojina: in some cultivars they are equally susceptible, whereas others show less infection on pods and stems (Sinclair, 1982; Yorinori, 1980; Yorinori and Homechin, 1978). Different isolates or races of P. sojina may also differ in their ability to infect leaves and pods (Yorinori and Homechin, 1978).

Host-Plant Resistance

Control of frogeye leaf spot is best achieved by growing resistant cultivars. However, considering the variety of conditions in which soyabeans are grown in different countries this may not be possible.

In Brazil, only cultivars that are resistant to P. sojina are released. In order to make this possible, all the breeding lines are tested at the local stations or the advanced lines are sent to the National Soybean Research Center at Londrina, Paraná, and tested in the greenhouse or in the field.

Most races have a wide geographical distribution and the same cultivar may be the host for many races. Although several races have been identified in Brazil, breakdown of resistance has not been frequent. Cultivar Santa Rosa, released in 1967, was resistant to frogeye until 1988 when race Cs-15 was developed. Cultivar Hardee, released in 1965, became susceptible in 1976. Currently, a new race (race 23) isolated from cultivar Doko, from the State of Goiás, is being tested on the Brazilian commercial cultivars (JT Yorinori, EMBRAPA-CNPSo, Brazil, personal communication).

Inheritance of resistance to P. sojina
Inheritance of resistance to the American races 1, 2 and 5 was studied by the authors who identified them. Resistance to race 1, found in cultivars Lincoln and Wabash, is controlled by a single major dominant gene designated Rcs1 (Athow and Probst, 1952; Probst and Athow, 1958). Resistance to race 2, found in cultivar Kent, is controlled by a major dominant gene and was designated Rcs2 (Probst et al., 1965). The resistance to race 5, observed in the cultivars Lincoln and Davis, is conditioned by two independent dominant genes at different loci (Phillips and Boerma, 1982). The resistant gene in Lincoln is the same as that previously identified by Athow and Probst (1952) and the gene in cultivar Davis was designated Rcs3 (Boerma and Phillips, 1983). Different dominant resistant genes to race 5 were also found in the cultivars Lee, Ransom and Stonewall and they were nonallelic to gene Rcs3 found in Davis (Pace et al., 1994).

Three independent dominant genes for the Brazilian race Cs-4 were found, one in cultivar Davis (gene Rcsa 3), one in cultivar Paraná (Rcsb 3) and a third in cultivars Santa Rosa and BR-27 (Cariri)(Rcsc3). The genes Rcsa3 and Rcsb3 also confer resistance to race Cs-15. A single dominant gene resistant to race 7 in China was designated as HRCS 7 (Yang et al., 1995). Resistance to frogeye leaf spot fungus race 1 in Chinese cultivars 81-732 and Ozzie is conditioned by a single dominant gene (Zhang et al., 1990). A new race of P. sojina designated race H was identified in Tokachi district, Hokkaido, on cultivar Suzuhime (Shirai et al., 1994).

Breeding for resistance to P. sojina
The backcross method and the screening of resistant lines through artificial inoculations at the F2 to F4 generations in the greenhouse is the procedure used in the development of resistant cultivars at the National Soybean Research Center, Brazil. Later generations are further tested in the field across the country through yield test plots (Yorinori, 1989). The inoculum of P. sojina used in the uniform tests is a mixture of the most common races collected from all possible sites of soyabean production in the country (Yorinori, 1989).

From the uniform tests carried out at Londrina, it is now possible to know the reaction to P. sojina of each commercial cultivar grown in Brazil. It also allows for the detection of a new race on a previously resistant cultivar, as happened in 1987-1988 crop season, when cultivar BR-27 (Cariri) became susceptible to race Cs-15. Cultivar BR-27 (Cariri) released in 1987 in the state of Maranhao, was the result of the cross [Bragg (3) x Santa Rosa] x (Bragg (2) x IAC 73-2736] where cultivar Santa Rosa was the source of resistance to frogeye leaf spot. The breakdown of resistance in Santa Rosa represented a considerable drawback in the effort to develop cultivars resistant to P. sojina for northern Brazil. Many of the cultivars that were resistant to the previous races became susceptible to race Cs-15. This also shows the vulnerability of soyabeans when grown under conditions favourable for a variable pathogen such as P. sojina (Yorinori, 1989, 1994).

When there are no facilities for artificial inoculation, field screenings of breeding lines can be performed by interplanting susceptible cultivars using seeds from naturally infected plants. If conditions are favourable for disease development, frogeye leaf spot should develop from inoculum produced on germinating infected seeds (Root, 1987).

A method of screening for resistance to P. sojina based on a disease index calculated from the affected leaf area has been proposed (Veiga and Kimati, 1976). For both field and greenhouse screening, it is essential to have susceptible cultivars as check plots for monitoring the efficiency of the inoculation and to compare the severity of disease development among breeding lines.

Sources of resistant genes for races of P. sojina occurring in the USA were reviewed by Tisselli et al. (1980). Some genotypes are resistant to specific races and are susceptible to others, but many are resistant to all races. Greenhouse inoculations on 156 cultivars using P. sojina race 5 in 1991 and race 4 in 1992 showed that less than 40% of the cultivars were susceptible to both races (Plopper et al., 1994). Several varieties have been released in the USA: Asgrow 7986, Buckshot 603, M82-722611, Wilstar 790, Hartz 7126, DPX 2106, Delta Pine 497 and S72-60 (Boquet et al., 1987), Carver (Weaver and Rodriguez-Kabana, 1995), Maxcy (Shipe et al., 1995), Doles (Boerma et al., 1994), TN 6-90 (Allen et al., 1993), Buckshot 723 (Harville, et al., 1993), Haskell (Boerma et al., 1994), Brim (Burton et al., 1994), Cook (Boerma et al., 1992), Crockett (Bowers, 1990), Stonewall (Weaver et al., 1989), A6785 (Shannon, 1989), Pella 86 (Fehr et al., 1987), Sherman (McBlain et al., 1987), Braxton (Goodin and Hinson, 1980), Pomona (Nickall and Schwenk, 1975), and Jupiter (Hinson, 1972). A genotype (PI417479) that is resistant to P. sojina race 2 was also resistant to stem canker (Diaporthe phaseolorum. var. caulivora) and to race 1 of Phytophthora megasperma f. sp. glycinea) (Brown and Minor, 1986). Several breeding lines were selected as resistant to frogeye leaf spot in India (Gupta et al., 1994), China (Ma et al., 1993; Qi, 1988; Shao et al., 1988; Shu et al., 1989) and Nepal (Chaudhary and Shanmugasundaram, 1987; Joshi, 1989).

For information on Brazilian cultivars with resistance to all races of P. sojina known to date and to stem canker (Diaporthe phaseolorum var. meridionalis), see Yorinori (1994) and EMBRAPA-SOJA (1996)

P. sojina is capable of causing large yield reductions. The greatest damage affects late-maturing cultivars (Lehman, 1928).From 1966 to 1968, yield losses in the USA varied from 17 to 21% (Laviolette et al., 1970). In 1991 and 1992, comparing susceptible and resistant varieties, losses varied between 8 and 43%, respectively (Plopper et al., 1994). Since 1952, in the midwestern states of the USA, frogeye leaf spot has decreased in importance and almost disappeared (Bernard, 1985; Yorinori, 1980). However, it is still common in the southern states, causing occasional damage on susceptible cultivars. The decrease in the incidence of the disease is attributed to the use of resistant cultivars and unfavourable weather conditions (Bernard, 1985; Phillips and Boerma, 1981). In 1978, a new race of P. sojina (race 5) was detected in Georgia, causing severe damage on the newly released cultivar Gasoy 17 and on Bragg (Phillips and Boerma, 1981).From 1971 to 1975, frogeye leaf spot caused severe damage (frequently crop losses of 100%) in Brazil. From 1972 to 1975, the number of susceptible soyabean cultivars grown was reduced from more than 80% of the total to a few hectares. In the 1987-88 growing season, an estimated half a million tonnes of soyabeans were lost to frogeye leaf spot in the west-central region of Brazil. The disease was favoured by an unusually wet season and because more than 60% of the area was planted with the susceptible cultivars Doko, EMGOPA-301 and Tropical (Yorinori, 1989).Frogeye leaf spot of soyabean is a potential threat in Nigeria, Côte d'Ivoire and Cameroon, where soyabean is being introduced for commercial production (Root, 1987). A recent outbreak was reported in Zimbabwe (Levy et al., 1996). Yield losses estimated at three locations in Nigeria ranged from 14.4 to 30.9% (Wala and Atiri, 1994). Studies in Nigeria showed that planting delayed until after 1 June was associated with a higher incidence of disease, with losses of up to 31% (Akem and Dashiell, 1994). In another study, the yield reduction between sprayed and unsprayed plots varied from 2.5 to 58.8% (Dashiell and Akem, 1991).From 1981 to 1994 in the Jiamusi region, Heilongjiang province, China, the relationship between epidemiology of soyabean frogeye leaf spot disease and meteorological conditions were studied using factorial and stepwise regression analyses. Primary mathematical models of disease forecasting were developed with the historical fitness of 92.9-100% for the two methods respectively. Positive linear correlations were found between the yield loss and disease severity on leaves, pods and seeds of resistant and susceptible cultivars (Huang et al, 1998).