Resistance of rice cultivars to Pyricularia oryzae in Southern Spain

The susceptibility of 14 commercial rice cultivars to Pyricularia oryzae was tested under f ield conditions agronomically appropriate for natural infection to occur. The first symptoms of infection were detected on the leafs when the crops were at the medium tillering stage. Generally speaking, the incidence of infection for each particular cultivar was similar in the 2002 and 2003 seasons and at the two trial locations (the Sartenejales and Casudis estates in the Province of Seville). The pathogen infected 80-100% of ‘Maratelli’, ‘Baixet’, ‘Thainato’ and ‘Galatxo’ plants. Differences were seen among the cultivars with respect to the parts of the plant affected. ‘Baixet’ and ‘Maratelli’ were the most susceptible cultivars, followed by ‘Thainato’. ‘Ullal’ was the most tolerant, followed by ‘Marisma’, ‘Puebla’, ‘Maso’, ‘L-202’, ‘Susan’, ‘Doongara’, ‘Fonsa’ and ‘Jacinto’; these last six cultivars showed medium disease severity according to the scale used in this work. Further studies on the interactions between different strains of P. oryzae and rice cultivars are underway. Additional key words: blast, Oryza sativa, varietal susceptibility.

'Rice blast', caused by the fungus Pyricularia oryzae Cav. is the most important disease of rice worldwide, both in terms of its distribution (Anonymous, 1968;Pans, 1976) and the damage it causes (Ou, 1972(Ou, , 1980)).Its great destructive potential can cause serious yield losses; controlling this disease is therefore one of the main goals of rice growers.
The presence of the disease in Spain was first mentioned in 1968 (Anonymous, 1968;Benlloch, 1975), and in 1978 it was reported in the paddies of the Marismas del Guadalquivir (Marín-Sánchez and Jiménez-Díaz, 1981).The aetiology and importance of P. oryzae disease was studied by Marín-Sánchez (1979), who reported Pyricularia infections to occur all over the Marismas rice area.These infections affected all the plant organs and caused serious yield losses when the climatic conditions where favourable to the disease; over 90% of the plants were affected.In 1997, rice blast seriously affected all the cultivars in this area; yield losses reached 15% -in economic terms some € 10 million were lost.
Pyricularia oryzae can cause damage to any aerial part of a rice plant, although the leaves and panicles (necks) are the most commonly affected organs.Leaf infection reduces the photosynthetic area of the plant, which can lead to its death.Panicle infection is that which causes the most important economic losses via yield reduction (Roumen, 1992).
The severity of the damage inflicted differs depending on the part of the plant affected and the cultivar.The use of resistant cultivars has been the cheapest and most effective way of controlling this disease, however, the high epidemic-causing and evolutionary potentials of this pathogen have led to plant resistance being overcome via the emergence of new fungal strains.In addition, the different types and degrees of resistance of the different cultivars, and the influence of environmental factors (Xia et al., 1993), affect the expression of resistance (Ou, 1980).
Currently, nearly all commercially grown European rice cultivars are susceptible to the fungus, yet there is little scientific information about the resistance of commercial cultivars to blast under European field conditions.
The aims of this study were: a) to describe the disease symptoms caused by P. oryzae in rice growing in the Marismas del Guadalquivir, and b) to compare the resistance of 14 commercial cultivars of different origin to natural P. oryzae infection under field conditions.

Material and Methods
All assays were undertaken at the Sartenejales (La Puebla del Río, Sevilla; 850 ha, 37º 22' N, 6º 10' W) and Casudis (Los Palacios, Sevilla; 2,400 ha, 37º 20' N, 6º 13' W) estates, both of which have clayey soils, during the 2002 and 2003 rice growing seasons.The altitude of both estates is just 3 m above sea level.The tillage and agronomic practices selected were those that suited P. oryzae infection (late seeding date, high plant density and high N-fertilisation rate).This allowed the disease intensity (Teng and James, 2002) associated with the different cultivars to be determined.
Fourteen commercial cultivars (belonging to both the Japonica and Indica groups) of unknown resistance to the pathogen (Table 1) were seeded in independent seedbeds between June 4 th and 10 th of 2002 and 2003.All these cultivars have early cycles and are adapted to the conditions of the Mediterranean climate.The plantlets were transplanted to plots at the two sites 30-35 days later (between the 9 th and the 13 th of July).These plots consisted of three rows (10 m long) 20 cm apart; the plants were spaced 25 cm apart.The experiment had a random block design with three replicates.Four additional rows were sown with 'Baixet' (known to be very susceptible to the pathogen in the conditions of the Marismas) and two more with 'Maratelli', a cultivar internationally recognised as susceptible to P. oryzae (Roumen et al., 1997).These, together with five additional terminal rows of 'Baixet' that surrounded the trial area, acted as an inoculum source.
Bearing in mind that in this area the first disease symptoms in previous years had appeared around mid July, samples (30 leaves, nodes, panicle bases and ligules per plot, depending on the phenological stage of the plants at the time of sampling) were taken and examined for infection every 10 days from July 15 th onward.These were packed individually by cultivar and date.At the end of the crop cycle, 30 additional plants per plot were sampled to determine the degree of disease in each tiller.The pathometric variables studied for each cultivar were incidence (percentage of affected plants) and severity (level of disease caused by the pathogen).Severity was studied by measuring the total plant surface affected and the number of affected nodes, ligules and panicle bases.The percentage infection of the whole plant, i.e., the weighted average of all the considered parts (adjusted percentage infection), was then calculated.This took into account the repercussion that each plant part might have on the grain yield if infected; for this weighting, the disease severity value for the ligule was multiplied by 1, that of the nodes and damaged leaf surface by 2, and that of the panicle base by 3.
The results were analysed by ANOVA using Statistix v.7 software (Analytical Software, USA).Least significant differences (LSD) were calculated (significance was set at P = 0.05).When the range of percentages exceeded 40%, arcsine x 1/2 transformation was used (Little and Hills, 1999).

Disease symptoms
The first symptoms of P. oryzae infection, whitish or green-greyish spots with darker greenish edges, appeared on the leaves in all cultivars when they were in the mid tillering stage.As the damage developed, the spots gradually became whitish-greenish in colour with brown-reddish necrotic edges (Fig. 1A).The spots were of different shapes although frequently ovalelliptic with the extremes more or less pointed and facing the same way as the veins.
When sporulation occurred, the centre of the spots turned ash grey due to the presence of conidia and hyphae.On average, the lesions were less than 1 cm long when the crop was in the plantlet stage, and reached 3 or 4 cm by the jointing stage.
In the culm (nodes and internodes) and panicle (base, axis, branches and glumes), necrotic dark brown lesions appeared that sometimes reduced or cut off the circulation of the sap.Node damage (Fig. 1B) was much more common and harmful than at the internodes; this sometimes broke the culm either partially or completely.The damage caused by P. oryzae was frequently occasioned by dewdrops carrying the fungus sliding down the flag leaf and collecting at the collar and affecting the ligule (Fig. 1C).
Infection of the panicle base (Fig. 1D) was usually the most destructive form of infection.As with node damage, this sometimes caused a reduction in the weight grain and even led to white, erect panicles with empty grains when the attack was early and severe.Infection of the panicle base and branches (axis, primary and secondary branches) and damage to the pedicels holding the spikelets were sometimes simultaneous, depending on the environmental conditions and cultivar.However, infections of the branches frequently appeared later, during the ripening phase.

Cultivar resistance
Table 1 shows the incidence of the damage caused by P. oryzae in the different cultivars, at the different estates, and in the different years of the study.Incidence figures varied among the different cultivars but were generally constant for each at the two sites and for both seasons.Pyricularia oryzae infected between 90% and 100% of the 'Maratelli' and 'Baixet' plants.'Thainato', 'Galatxo' and 'Hispagrán' showed mean incidence levels of 89.96%, 82.23% and 74.33% respectively.A third group of cultivars including 'Jacinto', 'Doongara', 'Fonsa', 'L-202', 'Susan', 'Maso' and 'Puebla' suffered medium incidence (40-60%).'Marisma' and 'Ullal', with 20-40% of plants infected, suffered low incidence of infection.
With respect to leaf disease severity (Table 2), 'Baixet' showed the highest percentage of spots, reaching values in 2002 and 2003 of 26.97% (i.e., leaf area affected) and 24.77% respectively at the Casudis site, and 28.26% and 28.35% respectively at the Sartenejales site.These values were significantly higher than those reached by the remaining cultivars in the same year and in the same locations.'Baixet' was followed by 'Galatxo', 'Thainato', 'Hispagrán', 'Maratelli' and 'Jacinto', which showed only low disease severity (10-15%).The cultivars that showed the least affected leaf area were 'Marisma' and 'Ullal', with values of < 2%.
The cultivar that showed the worst nodal infection was 'Maratelli' (significantly different compared to the remaining cultivars; Table 3).This was followed by 'Baixet', 'Thainato', 'Galatxo'and 'Hispagrán', which showed values ranging between 20 and 40% (i.e., of nodes infected).'Maso' and 'Ullal' were those that showed the least node susceptibility (or greatest node tolerance) to the pathogen.

Discussion
Despite the low incidence of P. oryzae in recent years in the Marismas del Guadalquivir, the incidence and severity of infection under the trial conditions provided enough descriptive and comparative data to determine the susceptibility of the different cultivars.
'Baixet' and 'Maratelli' were the most susceptible.With respect to leaf and panicle base infection, 'L-202' was more susceptible than 'Doongara', and more susceptible than 'L203' and 'L204' which were tested by other authors in California (Greer and Webster, 2001).However, 'Doongara' was more susceptible than 'L-202' to node and ligule infection.Node infections did not usually completely stop the flow of sap to other parts of the plant, and were not as destructive as infections of the panicle base; the latter usually caused empty grains and yield losses.Both cultivars have a similar genetic origin, an early cycle, a high resistance to lodging, are long grain varieties, and both are very productive (Aguilar and Grau, 1996), although 'Doongara' is somewhat more productive and is widely used in the Guadalquivir Valley.'L-202' has a slightly longer grain than 'Doongara' and is widely used in other countries.
The most resistant cultivars to the pathogen were 'Ullal' and 'Marisma'.'Ullal' has a medium grain and a short growth cycle under the Mediterranean climate; its grain yield is high and the plants are quite short.However, due to its high vitrosity, its market demand is reduced.'Marisma' stands out for its large grain size and culinary quality, it has a good agronomic performance, provides high yields, and is very interesting from an industry standpoint (Aguilar and Espinosa-Ruiz, 2003).
The results obtained in the present study may be useful when assessing the suitability of commercial cultivars for planting in areas where there is a risk of P. oryzae infection.In addition, the cultivars found to have a low susceptibility to P. oryzae could be selected as parentals in breeding programs.
Studies on the different strains of P. oryzae and their interactions with different cultivars are currently underway.

Figure 1 .
Figure 1.Symptoms caused by Pyricularia oryzae in rice: A, spots on leaves; B, node damage; C, ligule lesion; and D, panicle base damage.

Table 1 .
Incidence of P. oryzae infection (% damaged plants) by cultivar, site and season

Table 2 .
Percentage leaf area infection caused by P. oryzae per cultivar, site and season

Table 3 .
Percentage node infection caused by P. oryzae per cultivar, site and season

Table 4 .
Percentage ligule infection caused by P. oryzae per cultivar, site and season

Table 5 .
Percentage panicle (neck) infection caused by P. oryzae per cultivar, site and season

Table 6 .
Whole plant infection severity (taking into account the weighted data for each plant part) caused by P. oryzae per cultivar