Resistance testing of several genotypes of Kamba Local Paddy (Oryzae sativa L.) to leaf blight caused by Xanthomonas oryzae pv. oryzae

One of the important diseases of paddy plants is bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Xoo). Kamba is one of the local paddy plants in Central Sulawesi’s germplasm collection that is superior in origin from the Lore (Bada, Napu and Lindu). Until now, no scientific testing has been conducted on the resistance of local kamba paddy to leaf blight. This study aims to compare the level of resistance of several kamba paddy genotypes to leaf blight caused by the bacterium Xoo This research was conducted at the Disease Laboratory and Plant Pests and Diseases Gauze House, Faculty of Agriculture, Tadulako University, Palu. Stages of research implementation include propagation of Xoo, Preparation of test plants, and Transmission of Xoo. Observation variables include symptoms on plants, incubation period, disease severity and level of resistance to Xoo. The results showed that Paddy cultivars Kamba Bulili Kasar and Kamba Bulili Halus showed resistance reactions to Bacterial Leaf Blight (HDB) caused by Xoo bacteria. Both paddy cultivars also showed a disease incubation period of 6 days, longer than the susceptible comparison varieties.


Introduction
Central Sulawesi is very important as a genetic source in breeding and as an effort to maintain the identity of local paddy so that it does not become extinct.Kamba paddy is one of the local paddy plants in Central Sulawesi's germplasm collection that is superior in origin from the Lore plains (Bada, Napu and Lindu).The paddy has a fluffy taste, pure white color, distinctive aroma, and has a good shelf life after being cooked into paddy so that it is still cultivated by the local community, there are several types of local kamba paddy genotypes spread across the Lore plains [1,2].
In paddy cultivation efforts, there is always the problem of pest and disease attacks.One important disease in paddy plants is bacterial leaf blight caused by Xanthomonas oryzae pv.Oryzae (Xoo).This disease spreads throughout Indonesia and causes yield loss [3][4][5][6].The attack of this disease can reduce the quantity and quality of paddy produced because it can attack at all phases of paddy growth [7].
Symptoms caused in young stadia of paddy plants due to Xoo attack can be in the form of wilting (crackle), while when attacking at the stadia of tiller formation, flowering, and fruit ripening produce blight symptoms.This pathogen also causes damage to panicles and paddy grains, so that paddy seeds can be a source of HDB disease transmission in the field.Plant damage and yield loss of paddy by this disease can reach 15-80%, depending on the plant phase when the disease occurs.Yield losses caused by HDB in India reached 6-60% and in Japan reached 20-50% [8][9][10].The use of resistant varieties to the dominant pathotype in a particular area is one way to control Xoo.Disease control using resistant varieties is an effective, cheap, and easy control as long as resistant varieties are available in the community [11].For this reason, it is necessary to test local Kamba paddy against one of the diseases in paddy plants so that in the release of local varieties its characteristics can be known.This study aims to compare the level of resistance of several genotypes of Kamba paddy to leaf blight caused by Xoo bacteria.

Methods
This research was conducted at the Laboratory of Disease and Plant Pests and Diseases Gauze House, Faculty of Agriculture, Tadulako University, Palu.

Propagation of Xoo bacteria
Xoo bacterial isolates were obtained from the Subang Paddy Center, West Java.The bacteria used were propagated in the Plant Disease Laboratory by propagating using Nutrient Agar (NA) solid growth media with the composition of beef extract (Difco) 3 g.Peptone 5 g, agar 15 g, distilled water 1000 ml.The bacteria were spread evenly on the entire surface of the medium.Bacteria were incubated in an incubator for 2-3 days.Suspected pathogenic bacteria were re-isolated on new NA medium by scraping with an ose needle.Incubated for 2-3 days the pure culture of bacteria is isolated on NA medium for propagation.

Preparation of test plants
Kamba local paddy seeds of several genotypes namely Bulili coarse, Bulili fine, (obtained from Lore/Napu plain paddy plantations), Carkrabuana variety (susceptible control) and IR 64 (resistant control).Each paddy genotype was tested with 20 plants, resulting in 100 plant populations.Media that is ready, put into a screen house and given water so that it reaches field capacity.Planting media that has been ready, then planted paddy seeds that will be tested for resistance as many as 100 plants.

Transmission of Xoo bacteria.
Xoo bacteria that have been propagated to a sufficient number for further experiments carried out the dilution process in order to obtain the desired population density.Test plants that were 60 days after planting (HST), inoculation of Xoo isolates was done by Clip-method, i.e. the tip of the paddy leaf was cut approximately 2-3 cm with scissors that have been dipped in Xoo suspension with a bacterial population density of 10-9 CFU's/1 ml obtained from the dilution results.The leaves were cut as many as 3 leaflets on each plant.

Symptoms on the plant.
Observed by looking at changes in morphological symptoms both changes in shape, size and color from normal conditions to symptoms.

Incubation period
Performed by calculating the length of time required by Xoo bacteria that have been inoculated on healthy plants until the first symptoms of attack.

Percentage of disease severity
Observed every day since the plant was 1 day after inoculation until the last week.The observation data were then analyzed with the disease intensity formula, which uses the analysis method with the formula [12]: The criteria for resistance to leaf blight were classified based on the leaf blight index with the following categories: Resistant = 0-3; Somewhat resistant = 4-6; Susceptible = 7-9.

Disease Incubation Period
The results of the observation of the disease incubation period carried out by observing the lesions that first appeared are shown in Table 2.

Treatments average
Chakrabuana variety 0a Kamba Bulili Kasar variety 6.08b Kamba Bulili Halus variety 6.16b IR 64 variety 3.91c The observation of the disease incubation period showed that IR 64 paddy as a susceptible variety used for positive control showed positive results with the fastest incubation period with an average of 3.91 days.Meanwhile, both Kamba Bulili Kasar and Bulili Halus paddy cultivars showed a longer incubation period, with an average of 6.08 for Kamba Bulili Kasar and Kamba Bulili Halus slightly longer, with an average of 6.16 days.

Disease intensity
The value of variance showed that both Kamba paddy cultivars showed positive resistance to bacterial infection of Xanthomonas oryzae pv.oryzae.The average disease intensity is shown in Table 3.The 5% BNT test showed that the Kamba Bulili Kasar and Kamba Bulili Halus paddy cultivars showed a very low average disease attack intensity of 0.08 and 0.05 which was significantly different from IR64 paddy which showed the highest average of 17.6.Meanwhile, the negative control with distilled water did not show any results.

Figure 2. Blight symptoms 3 weeks after inoculation
Within 3 weeks after inoculation of Xoo bacteria to the leaves, blight symptoms were clearly visible on the leaves, namely curled leaves and gray lesions.These symptoms appeared clearly on the IR 64 variety which is a susceptible variety to HDB.Measuring the length of blight on the leaves is done by measuring the lesions that dry from the edge of one leaf to the edge of another leaf.Thus, lesions that only appeared on the edge of the leaf were not counted.

Discussion
Plant disease is one of the main problems in paddy cultivation because it is an obstacle in increasing national paddy production in Indonesia.The use of disease-resistant varieties is a good solution for paddy cultivation because it is an environmentally friendly paddy cultivation practice, reduces the risk of pest resistance, and even contributes to an increase in national paddy production by 56% [13].The bacteria used in this study was Xoo strain IV.The results showed that the Kamba Bulili Kasar and Kamba Bulili Halus paddy varieties showed a very significant resistance reaction to the disease attack.
In addition, the incubation period of the two Kamba paddy cultivars was significantly different, which was 6 days compared to the IR64 variety which was classified as susceptible and had an incubation period of only 3-4 days.This suggests that the use of these Kamba paddy cultivars can be effective in cultivating disease-resistant paddy.Meanwhile, the IR 64 paddy variety used as a positive control showed susceptible results as it should.This is because IR64 paddy is classified as susceptible to HDB disease with a very short incubation period [14].
Based on observations of disease attack symptoms on the leaves after the incubation period, HDB symptoms were found, starting with brown line spots around leaf wounds, then dry spots expanding whitish gray and slightly curled leaves.These symptoms also make the leaves become rather brittle and easily crushed.This can cause paddy productivity to be reduced because damaged leaves cause the photosynthesis process to not be maximized [15].
The HDB pathogen can enter through the hydatode, which is the place where gutasi evaporation occurs at the edge of the leaf.That is why lesions can be seen on the edge of the leaves at the beginning of the disease symptoms.However, the most potential entry point for bacteria is leaf wounds, especially new wounds compared to old wounds.Later, disease progression characterized by the development of whitish-gray lesions can be influenced by sunlight, disease virulence, and the genetics of the strains used [16].
This resistance could be due to the fact that both Kamba Paddy cultivars have resistant Xa resistance genes that cannot be broken by Xoo strain IV bacteria.This is because Xoo strain IV bacteria have virulence that can break seven types of resistance genes, namely Xa-1, Xa-2, Xa-4, Xa-7, Xa-10, Xa-11, and Xa-14.This causes pathogenic bacteria to have specific behavior in invading their host plants, because one type of bacteria can infect and multiply only in certain hosts [3].
In addition, the two Kamba Paddy cultivars also showed leaf morphological characteristics associated with resistance to HDB disease based on research from Rohaeni and Yuliani [17].These two genotypes have the same characteristics of green leaf neck color, white leaf tongue color, white/transparent leaf ear color, and downy leaf surface.This leaf morphology has been shown to have a positive correlation to HDB disease resistance.It is thought that a plant becomes resistant to HDB disease due to the presence of phytoalexin compounds as a result of host-parasite interactions whose function is to inhibit bacterial development.The accumulation of phytoalexins in high concentrations can limit the area of pathogen infection on the leaves.This causes leaves treated with bacterial suspensions to dry only slightly at the tip of the leaf [18].
It is also explained in the research that paddy plants have a resistant response to blast disease because they accumulate the phytoalexin compound sakuratenin which is a toxin for plant pathogens quickly after being infected with pathogens [19].Therefore, phytoalexin compounds are also thought to have an effect on inhibiting bacterial leaf blight in Kamba cultivar paddy.Research by Djatmiko and Fatichin (2009) reported that the generative phase of paddy plants is more resistant to disease attack compared to the vegetative phase.Meanwhile, the susceptibility of paddy plants to HDB disease in the generative period is caused by a decrease in the value of resistance if the leaves have been infected since the vegetative period.Therefore, testing plant resistance to disease would be better if done only once in the growing period, either during the vegetative or generative period.

Conclusion
Paddy cultivars Kamba Bulili Kasar and Kamba Bulili Halus showed resistance to Bacterial Leaf Blight (HDB) caused by Xanthomonas oryzae pv.oryzae (Xoo).Both paddy cultivars also showed a disease incubation period of 6 days, longer than the susceptible comparison varieties.

Figure 1 .
Figure 1.Early symptoms of bacterial blight Based on the early symptoms that appear, the characteristics of bacterial leaf blight are shown, namely brown lines around leaf wounds and gray blight lesions.

Table 1 .
Scale value of leaf blight disease in paddy