Morpho-physiological characterization of bacterial grain rot in rice seed from Gowa Regency, South Sulawesi

Bacterial Grain Rot caused by Burkholderia glumae is an important disease of rice. In South Sulawesi, this seed-borne pathogen has been detected in several places. There were some reports from the farmers regarding rice yield loss due to this disease. This study aimed to isolate and characterize morpho-physiologically bacterium causing grain rot of rice in the main rice-producing areas of Gowa Regency, South Sulawesi. Samples were obtained from 10 districts. Several grains of rice were isolated and grown in King’s B medium. Morphological characteristics were determined by the color, form, edge, and elevation of colonies. KOH solubility, catalase test, anaerobic growth, toxoflavin production assay, production of fluorescent pigment, and colony color on YDC medium were performed to determine physiological characteristics. 1 isolate from each area was found which has similar morphological traits to B. glumae. Colonies that were yellow, yellowish white, creamy white, and grayish white, with a circular form, entire edge, and convex elevation. The result of physiological characterization showed that all isolates tested positive for KOH solubility and catalase test as well. All isolates showed negative reactions for anaerobic growth, production of fluorescent pigment, and colony color on YDC medium. 5 isolates were able to produce yellow pigment (toxoflavin), so it can be concluded that all of the isolates that have been characterized morpho-physiologically are presumably confirmed as B. glumae.


Introduction
Burkholderia glumae is one of the most serious seed-infecting bacteria in many rice-producing areas, particularly in South Sulawesi, Indonesia [1].This bacteria causes grain rot on rice [2].This seed-borne pathogenic bacteria [3,4] causes yield losses ranging from 40% [4] and up to 80% in the worst case [5].Nowadays the disease was announced to have harmed rice farms in several regencies including Maros, Barru, Sidrap, and Luwu in South Sulawesi [6].
Due to this disease symptoms are similar to those abiotic factors such as water stress, heat disturbance, or other factors related to the environment, and biotic factors with various kinds of seedborne pathogens such as sheath brown rot disease, bacterial brown stripe disease, or damaged by insects such as rice ear head bug, it is frequently misdiagnosed [7][8][9].Bacterial grain rot disease was characterized by reddish-brown necrotic lesions that began at the base of the grains, severely diseased panicles that stayed erect owing to blank and also exhibited aborted seeds with a dark basal rot, and badly afflicted plants [7,10,11].Because it assaults the generative phase, this condition will be extremely harmful and arduous to control if it did not observe carefully.Therefore, characterizing the causal agent of bacterial grain rot is an important precondition for disease management.

Sampling
Samples were obtained in 10 districts in the main rice-producing areas of Gowa Regency, South Sulawesi using a non-probability purposive sampling method.Grain rot signs were seen on those samples.Samples were placed in plastic bags and labelled, then taken to the Plant Diseases Laboratory, Faculty of Agriculture, Hasanuddin University for further analysis.

Isolation
The rice grain samples were surface sterilized with 2% of NaOCl for 2 minutes then washed 3 times with sterile distilled water every 30 seconds to sterilize the pathogens on the surface [6].Isolation method according to Isnaeni and Masnilah [12] with modification. 1 gram of each sample was crushed and then 9 ml of sterile distilled water was added then incubated for 2 hours at room temperature so the bacteria could diffuse out of the plant tissue.A serial dilution of 10 -8 in sterile distilled water was made for each sample.Then, 50 μl of each sample dilution was streaked on King's B Agar medium and incubated for 48 hours at 33°C.

Morphological characterization
Testing the morphological characteristics of the colony which included color, form, elevation, and edge were the characterization procedures [13].The bacteria that were presumptive on B. glumae as described by Aflaha et al. [6], Coenye and Vandamme [14], and Singh and Vishunavat [15] were chosen to perform physiological characterization.

Physiological characterization
Physiological characterizations were used for the following test: KOH solubility, catalase test, anaerobic growth, toxoflavin production assay, production of fluorescent pigment, and colony color on YDC medium [16].

KOH solubility.
Bacterial isolates were tested by a solubility assay in KOH 3% solution to observe the gram reaction.Isolates were tested by mixing one loop of isolate and 1-2 drops of 3% KOH solution on object glass.Gram-positive if the mixture did not turn into mucoid, while gram-negative if the mixture turned into mucoid.

Catalase test.
Catalase test was executed by using 3% of H2O2 solution.1-2 drops of 3% H2O2 solution mixed with each ose of bacterial isolates.The positive result is indicated by the presence of gas bubbles, while the negative result showed no gas bubbles.

Anaerobic growth.
Hugh and Leifson's OF basal medium were used to distinguish between the aerobic and anaerobic breakdown of glucose.The tubes for aerobic and anaerobic fermentation were inoculated and one tube of each pair was covered with a 1 cm layer of 10% agar to create the anaerobic condition in the tube by inhibiting the flow of oxygen and the other tube exposed to the air.All tubes were incubated for 48 hours at 35°C.The aerobic result indicated by the tube without agar turned from greenish blue into yellow while the covered tube is not.The anaerobic result indicated by both tubes turned yellow.

Toxoflavin production assay.
All isolates were grown on King's B Agar medium and incubated for 48 hours at 37°C.The emergence of bright yellow color in the medium indicated the production of toxoflavin by the isolates.On the contrary, when it does not appear then the result is negative.2.4.5.Production of fluorescent pigment.Fluorescent pigment production was determined by the presence of glowing yellow-green or blue-green color of isolates in King's B Agar Medium when placed under a UV light.Otherwise, the result is negative.
2.4.6.Colony color on YDC medium.All bacterial isolates were grown on YDC (Yeast-extract Dextrose CaCO3) medium and incubated at 37°C.Positive reaction when the bacteria forms yellow with mucoid consistency and convex colonies, whereas negative reaction if it does not.

Sampling
From 10 different districts, 1 sample from each district was collected.There are 4 rice varieties grown by farmers.The samples were isolated from Pattallassang with Inpari 32 variety.Parangloe with Ciherang variety, as well as Pallangga, Bontomarannu, Somba Opu, Barombong, and West Bajeng.Ciliwung variety in South Bontonompo and Bajeng, and the last variety was from Bontonompo with Cigeulis variety.The rice grains collected are displayed in Figure 1.It showed necrotic lesions develop from the base of the grains and empty rice grains due to severe damage.The symptoms of B. glumae are small rot with brown edges on the panicles, hence causing empty grains [11,17,18].

Morphological characterization
Morphological characterization results of color, form, elevation, and edge of the colonies can be seen in Figure 2. Morphology characteristics of the isolates have different characteristics.It showed that the colors are white cream, grayish white, yellowish white, and yellow with a circular form, convex elevation, and entire edge.Colonies of B. glumae that appear on King's B Agar medium are circular, convex, and entire edge with grayish white, yellow, yellowish white, or creamy white [6,14,15].The difference in color from grayish white to yellow in King's B Agar medium is due to water-soluble pigments by B. glumae [14].Bacterial colony characteristics from the colony color showed a significant level of variation among the tested isolates.The differences in bacterial isolates indicated the diversity of bacteria in terms of their morphological traits [19].For more details, see Table 1.

Physiological characterization
Physiological characterization results of KOH solubility, catalase test, anaerobic growth, toxoflavin production assay, production of fluorescent pigment, and colony color on YDC medium are shown in Figure 3.For all samples are listed in Table 2.

KOH solubility.
The result of KOH solubility showed that all bacterial isolates belonged to the gram-negative bacteria group as indicated by the mixture turned into mucoid.B. glumae shows positive result for the KOH test which produce a slimy consistency in the bacteria so that they are included as gram-negative bacteria [12,15].Suslow et al. [20] said that gram-negative bacteria have a thin cell wall that breaks easily in high-alkaline solution.The rupture of the cell will release DNA molecules that are sticky threads.

Catalase test.
Positive reaction on all bacterial isolates were shown, as evidenced by the production of air bubbles.This meant that all bacteria were able to produce catalase enzyme which converts H2O2 to H2O and O2.Catalase test on B. glumae produces positive catalase [2,6].

Anaerobic growth.
The result of the anaerobic growth indicated that all the bacterial isolates tested were aerobic (oxidative).This indicated that none of the bacterial isolates could produce the enzyme that ferments glucose in aerobic conditions.Isnaeni and Masnilah [12] observed that B. glumae bacterium exhibit oxidative reaction, which is characterized by change in media color from blue to yellow in paraffin-free media.
3.3.4.Toxoflavin production assay.5 out of 10 isolates that were BmCh1, BSCw1, PgCh3, PlIn2, and SOCh5 could produce toxoflavin pigment by the emergence of bright yellow color in King's B Agar medium and the rest of them were not able.This indicated that half of all bacterial isolates strains were virulent, and the others were avirulent.The presence of yellow toxoflavin hue in B. glumae is a virulence agent that causes grain rot on rice.Some avirulent strains have been isolated from symptomatic rice grains [21,22].However, these strains could not generate toxoflavin [10].Joko [23] stated that toxoflavin is one of the main phytotoxins in rice which can suppress shoots and root development in rice seeds and induce grain rot in rice.

Production of fluorescent pigment.
The fluorescent pigment test result showed that none of the bacterial isolates produced fluorescent pigment when placed under a UV light.This is according to Karki et al. [21] that B. glumae bacterial isolates cultured in King's B Agar medium do not produce green diffuse pigments that fluorescent when exposed to a UV light.

Table 1 .
Morphological characterization of the bacterial isolates.