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show various changes in their physiology, such as respiratory rate, photosynthetic efficiency, amount of component substances, enzyme activities as well as water permeability of leaf cells (Ezuka and Kaku, 2000). Healthy leaves of the susceptible eultivara show higher photosynthetic efficiency than the tolerant cultivars, The photosynthetic efficiency itt water soaked and straw-yellow regions was lower than in the green region of the infected leaves and lower in the susceptible cultivars than in the tolerant cultivara, Chlorophyll a and b and total chlorophyll contents were higher in the green region than the water soaked and straw- yellow regions and the ratio of chlorophyll coute were higher in the green and watersoaked regions of the susceptib1 eultivars than the tolerant cultivara (Philip and Devadath, 1981). It was found that in susceptible leaves, oxygen uptake increased with the increase of bacterial cells as compared to that of healthy tissues after the appearance of initial symptoms (Watanabe and Asaumi, 1975). Moreover C assimilates were not accumulated at the inoculation sites, while accumulation of assimilates was remarkable in the border parts of fresh yellow streak lesions adjacent to healthy tiSaUC5 (Watanabe et at., 1980). Permeability changes were studied in rice leaves inoculated with bacterial blight pathogen by measuring leakage of electrolytes and C assimilates and was found that living bacteria enhanced the permeability of rice leaf pieces (Kohno et al,, l98 In leaves of bacterial blight infected plants, the coctentS of reducing and non-reducing sugars, crude starch, ammoniuns nitrogen, total phosphorus, lipid phosphorus, nucleic acid phosphorus and insoluble phosphates increased, whereas the amount of total nitrogen water soluble protein, and water insoluble protein-nitrogen decreased. Among them, the increase of crude starch, lipid phosphorus and nucleic acid phosphorus was assumed to be mostly due 10 the bacterial body of the pathogen present in the sampleS analyzed (Misawa and Miyazaki, 1973). As far as the activity of enzymes is concerned it was observed that the activities of celluI invertase, xyiase, phosphataae, 8 cs-amylase, pectinase, lipase, lecithinaae and proteaae increased in diseased tissues as compared to healthy ones. The activation was more remarkable its the early stage of infection than in the later stage except -arnylase and cellulose (Miyazaki es of, 1976). The activity of ?eroxidase increased nsarkedly a the lesion enlarged in susceptible leaves (Akutsu and Watanabe, 1978). ETIOLOGY OF BACTERIAL BLIGHT DISEASE IN RICE The bacterial nature of leaf blight in rice was established by Japanese scientists itt tlse early 1920s (Mew at aL, 1903). The causal bacterium is Xanthornonas oryzsw pv. otyzae. Initially X. sn-yeas pv. oryzas was considered as one of the pathovara of Xrsnthornoncss campSsrris till 1992. Afterwards, it was reclassified as Xa erycac pv. o,yzue. According to the descriptiop of lshiyama (1922), X oryzae pv. oryzae is short and rod
capsule-like material composed of hstra-polysaccharide. o pv. 1962).yzae develops yellow. 2002). while that for dried cells is 10 minutCs at 56°C (Tag and Mizukattii. succinic acid and fumaric acid (Noda at al. The bacterium is aerobic. ersdoplasnsie reticuis or mesosomes were found. Nuclear materia of fibrillar appearance. 1959). 4941439 bp. Its genome consists of a single. 3-methylthio acid.shaped. glucose. They were identified as trans acid. 0. circular. sucrose was the most favorable carbon source. galactose and ms1to Suctinic acid was also good carbon source among the organic acids tested. in most cases.7 urn in host. Therefore.0 X 1. and a cytoplasm membrane composed of a continuous Uttli membrane was present slung the inside of this cell wall. BPS is regarded as a vimience determinant (Shen and Ronald. with round ends.. snanose. This material was assumed to serve as a protectant frosts unfavorable conditions such as drought (Mizukami. o. but no mitochondria. Hodno (1973) examined the ultrastructure and showed that the bacterium was surrounded by a relativoly electron transparent cell wall. manose. glucuronic scid. The extracellular polyssecharide (EPS) was reported to be composed of glucose. produces a small amount of hydrogen sulfide. Various colony type varisnts of X otyzae pv. tiglic acid. and arabitol (Misaki at al.0 The temperature range is 5-40°C with the optimum of 26-30°C. isovaleric acid. Gram negative. 1962).0-7. the mutant types showed atlenuated virulence or avirulence as . ruannose. 1922). digests milk without coagulation. The pH range for bacterial growth is with the optimum of 6. glucuronolactone. phenyl acetic acid. oPyzae were obtained due to naturai or induced mutation in artificial culture. followed by glucose. oryzrse. Various strains of X osyzae pv. The thermal death point for multiplying cells is 10 minutes at 53°C under wet condition. but not ammonia or iridole. does not reduce nitrate or methyletse-blue. orlzae seCtCte certain toxic substances. Surface of the bacteria is covered by a viscous. reddeess litmus roOk. glucurortic acid and glucuronolactone was also isolated (Atsgadi. 1980). with monotrichous flagellum of 68 cm. 1955). Seven substances toxic to rice seedlings were isolated from cultured cell suspension of X. storage and culture. does not liquel gelatin. and polysome hke sbrjctssres were observed in the cytoplasm. o have been found to carry one or more indigenous plasmids with molecular weights of about 20. A high molecular weight polysaccharidu made up of monosaccharides. 1978). smooth and viscous colonies on potato semi synthetic agar medium (Wakitnoto. long-terrr. and does not produce gas from sugar (lshiyama.3 X l0 1021 X iO dalton. X oryecis pv. X oryzoe pv. aerobic and non-spore forming.. circular chromosome. Spontaneous loss of virulence associated with reduction of EPS production has been observed it. ribosoms. Thc growth on the medium requires carbon and nitrogen sources.8-1. According to Watsnabe (1963).
and the disease is generally tuore severe in the wet season than in the dry season (E and Kaku 2000). i9l I) due to increase in number of leaves arid prolonged vegetative stage. Therefore it is natural that the disease is hardly seen itt upland rice tiniess the land is inigated or flooded by heavy rainfall (Kuwatsuka. Soil type appears to be a factor affecting the incidence. as th dIsease was found to be severe in rice plants grown in sandy loam.. arid typhoon during th rice-growing season (Bokura. The advantage of vascular infection to the pathogen is that water and nutritional feetors essential to growth of the bacterial pathogen are rich in the vascular system and the pathogens are less influenced by boat resistance reaCtions (Ezuka sod Kaku. temperature is the factor limiting the period of bacterial blight incidence to warmer seasons. clay. But a close relationship was observed between tolony types and serotypea of the mutant strains of X. humidity. oryzar pv. Haw most of the general bacteriological properties were found to be similar to those of the wild type strains. However there is remarkable alteration of the wet and dry seasons. optimum temperature for bacterial blight lesion development is reported to be 25-30°C. In temperate regions. while the symptoms hardly appear at a I temperature of 17°C (Mukoo et a2 1957). flood. However in india. topographic arid soil conditions. in tropical regions. X. temperature is high enough for disease development throughout rh year. or clay loam alluvium soils. ECOLOGICAL FACTORS AFFECTING THE OCCURRENCE OF BACTERIAL LEAF BLIGHT IMSEASE IN RICE The incidence arid severity of bacterial blight are influenced by various environmental factors. temperature. Consequently climatic conditions greatly influence disease incidence and development. oryzae pv. rainfall and nitrogen fertilization ace the moat itoportant factors affecting the occurrence of the disease (Ezuka aced Kaku 2000). 1959). o. 967). 1982). 1981). 1944). though phsge sensitivity of some mutants had changed (Tsuchiys at at. the bacterium cast multiply oniy irs xylem vessels. nitrogeC it most responsible for encouraging diaesse devclopment as reported by a large number of researchers (Bokura. oryzas is a typical vascular pathogen. and cultural practices. IPil).yzaa (Choi at a!. They are divided into climatic conditions. Among vascular elements. 1978). The multiplication and translocatiori of the pathogen are limited to the vascular system. and slight in the sandy csil adjacent to dune areas irs the Hokuriku area of Japan (Yoshimara. That is why the most important factors for disease epidemics in Japan are rainfall. Whereas. longer lesions wece produced on plants in lateritic soils than in sandy soil (Dath at at.compared to the wild type (Goto and Okabe. Among them. Fertilization is one of the most important eultural practices affecting the development of bacterial blight ire rice. 2000). It enters rice leaves through water ptsres or wounds and spreads into the vascular bundles. Bacterial blight is a typical water-bum disease because the causal organism is disseminated by irrigation water. Among the threc elements of fertilizers. As far as the temperature is concerned. .
4941439 bp circular chromosome. A total of 271 out of 478 protein coding sequences its the id sntifind IS elements of the Xøo genonse showed significant similarity to transposasea. Thus. ware also identified. Virulence and regulatorj genes required for bacterial pathogenicity are commonly found in pathogenicity islands (PAls) (Lee at a!. Many of the IS elements are located near strsin specific genes (Lee eta!. aayrae provid ss molecular genetic evidence that resistance in the interaction between X o pv. Isi addition. aryzae strain KACCIO33I. each consisting of two operons.genicity (hrp) gene cluster was identified in the X.. e. hrp-mutants cannot elicit the HR in incompatible interactions with resistant host plants. 1Slll3 IStl4. known as xanthan gum. The assembled sequence was found to be consistent with a singte. Genes encoding SRNAs that recogeize 54 codons were also found. auggesting that hrpX gene confers a means to evade the host defense response (Ezuka and Rake. oryzae genome that included 26 genes. RTX toxins are important virulence factors for a variety of psthogens. OREZAE The basic features of the genorne of Xonthomonas oryzae pv. oryeae pv. the RTX toxin genes found in the X oryzae pv. TNXG and TNX7) had been identified in another Strain of Xoo. 2005). The genes for two RTX toxins. indicating that these have played an important rote in hotiz gene transfer and also in iaternsl rearrangement of the genonse.7%. Cloning of ass avirulent gene from X o?yraa pv. Two separate sets of 23S-5S and I 6S rihosrunat RNA (rRNA) genes. The hrp genes see required for many bacterium-plant interaction phenotypes. . a. Cuttivar-/race-apeciuic interactions characterized by gene-for-gene cornplementarities have been proposed to be controlled by an avinslest (act) gene from the pathogen and a corresponding resistance R) gene in the host.MOLECULAR GENETICS OF XANTHOMONAS ORFZAE PY.yzae and rice follows a gene-for-gene mechanism and provides a means to investigate physiological and biochemical mechanisms of resistance (Ezuka and Rake. rtxA and rtxC. were identified in X oryzae pv. 2000). is produeed by the activity of the gum cperon products. 2005). genes has pleiotropic effects.yrae genotste. Most of the studies on molecular genetics of the bacterial blight pathogen so far have been concerned either with pathogenicity or productioc of polysaccharides. Five inscrtion sequences (1St 1 lT=TNX8. mutants are attenuated in their ability to colonize and initiate disease symptoms in host plants and cannot elicit the hypersensitive reaction (RH) in nob-host plants. The average G+C content was 63. The lu gene is essential for pathogenicity and a gain in the ability of Xrsntlwmortar to cause the hypersensitive response in their respective host plants. oryzae genome may also be virulent factors. inactivation of the . A Hypersensitive reaction and pathc. Most of the genome was coding sequence and contained 4637 Open Reading Frames (ORPa) predicted to encode polypeptides.. EPS play a critical role in facilitating adhesien of bacteria to the host surface during initial stages of plant pathogen interactions and disease development. a representative Korean race 1 strain are givers in figure 7. The extracellular polysaceharide (EPS). 2000).
X.RX collaborative program aiming sri the development of international differentials consisting of near isogenic lines (Ogawa at tsL.. TKM6. asyzae pv. Ia. Japan and India and identified It pathotypes which showed different virulence as compared ton those reported from Japan and IRKS. Noda at aL. EXISTENCE OF RACES IN THE PATHOGEN RIce cultivars are quite diverse in their gertetie traits. 1981). the collaboration has been focused on the Japan-IR. India. Thailand. its order to compare the pathogenic differentiation among different countrie an international set of differentials is desirable. osyzae for plasmids. (1992) screened 41 isolates of K. Reddy and Reddy (1989) tested 150 isolates from various parts of India for their virulence to six rica differentials. Rehrnan at a!. Nepal. He found that all isolates of X oiyzcse pv... were developed. For that purpose. Malaysia. LII and IV. and evaluated their physical and pathological properties. osyzae pv. Each country has its own differential system. the differential cultivars used for race differentiation are different depending on the country. and race lB out of . Mohiuddin and Kauffman (1975) reported from India that 24 Indian bacterial isolates were divided into eight virulence patterns based upon their reaction to seven differeritisi cultivars. Censpo Selak and Java 14. The plasmid was reitotsted from the inoculated rice plants. Therefore. The differential systems presented from different countries have an essential significance in the respective country. and identified three pathotypes. Amuthan at at. DV85.3 1< to 2 X tO dalton. Durgapal (1985) divlded Indian bacterial isolates into five pathogenic groups from I to V. indicating the stability of the plasmid and involvetuatst of indigenous plasmid in bacterial pathogeneass. osyrrrr. Ib. orysos pv. an international collaborative research project was commenced by IRRI irs 1977 with the participation of IP Rasigladosh. T65 and Taichung Native 1. vinalence of the pathogen also seems to be quite different depending on differ countries arid areas (Hcirino. 1966. six races have been identified on the basis of reactions with IRRI differential cultivara that contain different resistance genes. arid are quite different in their resistance depending on their derivations. Itt Philippines. 831. (1986) tested 13 indian isolates for their virulence to nine cultivars from IRKS. II. BJl. and the molecular weight of the plasmid was about 20.. Strains of X oryzae p osyzoe have been classified into many races based on their virulence on differential rice varieties.. lR24 Toyordshiki and Milyang 23. (1988). indonesia. on the basis of their reaction on four rice cultivars. IRS. This program was accomplished by Ogawa at a!. 1981). Pal rEal. However. (1993) t 122 isolates for their pathogenic variation using five iRRJ differentials and identified five races. Twenty-five isolates contained 1-4 plassnids beta single plasmid was detected inmost of the isolates. and three sets of differentials with single major resistance genes so far known arid with near-isogonic background of three eultivars. Gupta at a!. identified race I. 1991). Likewise. race LA. Ib. oozes harbored plssmids. race II. otyzse has also been reported to have one or more indigenous plasmids. China and Japan (Henna. investigated the plassnids in various strains of X. Since 1982. (1988). lR2O. Ia. II. Korea.
Choi St of.4 percent. respectively. (1977) tested 71 isalates for the virulence to Japanese differentials.5 and 0.6. Ill and V in the ratio sf79. 0. and one as race V. Horino et al. In Vietnam. (1979) tested 224 Korean bacterial isolates for their virulence to Japanese differentials. (1985) tested 201 isolates using newly selected Korean differentials. IV and V were somewhat different among the localities. The predominant races I and 11 were found in almost all the areas. If more isolates and more cultivars are tested in future. Yarnarnoto at of. Ill.7. Changes ir. and found three races. 33.213 isolates thorn various localities of Japan. Therefore . and the differential system would become much more complicated. Yun er of. 16. in the ratio of 64.0 per cent. in the ratio of 56. surveyed the distribution of races throughout Japan. On the other hand. reapectively. 259 were identified as race I. (1999) divided 52 bacterial isolates into six races tentatively named A to F. I. K3 and K4. among 427 isolates tested.9 and 1. Out of 120 isolates tested. respectively. More than 80% of the isolates were clasaified as race A. 18. GENETIC DIVERSITY OF THE PATHOGEN There are a number of factors for bacterial virulence. pathogenic variability occur rapidly because of faster reproduction and more frequently mutating ability of tise pathogen. IV and V in the ratio of 77. more groups of isolates and cuhivars might be distinguished. but genetic variation also exists in the pathogena. making them either avirulent or virulent against a certain host. Races A and B were identical with Philippine races 3 and 1. and identified four races. tested 625 isolates and found five races. The variation claimed to be contiguous is based on the fact that the lesion length caused by bacteria on certain varieties gradually increases from Japan to tropical Asia (Budde and Reddy. respectively. In Korea. II. Kl.3. In another study he recorded 27 races from 9 countries in South and East Asia on the basis of their pathogenicity to Japanese and Philippines differentials. 1(2. Yun et of (1984). a majority of9l were classed as race 1.4. on the basis of the reaction pattern to 18 rice cultivate including near isogenic international differentials. 128 as race I 36 as race II three as race IV. while the distributions of races II.3 per cent. 1978). and reported that. 1972). In Indonesia. and found four races. respectively. improvement of the differential system on the basis of genetic studies would be necessary (Ezuka and Sakaguchi. tested bacterial isolates collected from the Tokai-Kinki region of Japan for their virulence to Japanese differentials. These results showed that the dis of races in Korea was similar to that in Japsn. based on the virulence patterns against Japanese plus IRRI differentials.. while 21 and 8 were classed as race II and race III. In that case. The geographical distribution of bacterial groups or virulence groups referred to as races has been reported by a number of researchers.8. Horino (1978). (1983) reported that 25 iaotates out of 40 tested belonged to race A and 6 to race B. Ezuka and Horirto (1974). 3. I. 3 and 3 per cent. also using Japanese differentials. I IV and V.8 and 1. Nods at al.. II. 8. 13. 23.
In zone 3. Nawab Shah. Guiranwala. SURVEYS OF RICE ZONES OF PAKISTAN FOR EVALUATION OF BACTERIAL BLIGftT DISEASE Surveys of all rice growing zones of Pakistan were conducted at the mature grain stage during the year 2002 and 2003 Is zone 1. oryzae isolates were characterized using biological and molecular methods. Efforts are therefore needed to identify other getses required for virulence and to identi other genes required for virulence and to elucidate the concerted functions of all the virulence determinants (Shen and Ronald.it is essential to understand the variability in the psthogsu in relation to its virulence (Ezuka and Kaku. Sheikhupura. o pv. A aignificant number of X. Narowal. aryrae genes have been found to be involved in virulence. National Agricultural Research Center (NARC). areas of NVTFP such as lower Dir. including its prevalence. Gujrat.yzae pv. Dadu. Kasur and Okar were surveyed from 4 October to 4 November2002 and from 35 October to 1 November 2003. and genes encoding components of secretion systems for delivery of extracellular enzymes and virulent and avirulent factors into plant cells It is now clear that X o. genes for synthesis and regulation of other virulence and avirulence factors. 1-lafizabad. These include genes required for BPS production. th large teact of land on the west bank of the river Indus including the areas of Sindh such as . Jacobabad as well as few areas of Balochistan including Nasirabad and Usta Muhanunad were surveyed In case of Sindh survey was conducted between 31 . Bacteria were isolated from the samples and Xasthornenas oryzae pv. Changes in race/ pathotype atrucrure within the population may result from several factors such as genetic change (mutation or recoinbination in response to agricultural MATERIALS LUST) METHODS For the investigation of bacterial leaf blight diseaso of rice crop surveys of famier rice fIelds were conducted throughout the agroecological rite zones of Pakistan. Larkana. Swat and Malakand Agency were surveyed from 4 September to 13 September 2002 and from 14 September to 16 September 2003. 2000). Sheikarpur. Evaluation of the disease. Islamabad. Sialkot. Pakistan. incidence and severily in different rice growing areas was done in fields and leaf samples from each field were collected. 2002). orysae virulence is very complex and not yet completely characterized. The foliar samples were then brought c the Crop Diseases Research Programme (CDRP). Ic zone 2 the areas of Punjab such as Sargodha. Lahore.
For scoring of BB severity in field following scale was used. Prevalence was calculated with the help of following formula. starting ten (18) paces into field. 1996). FIELD BASED DISEASE ASSESSMENT AND SAMPLING Iii each disinct number of locations visited depended upon cropping intensity. On each location surveyed. planls were observed at 5 pOints along a diagonal transect. Percentage average lesion area of the fifteen leaves collected was taken for disease severity in each field. including the areas of Thatta and Badin were surveyed. Severity .August and 3 September 2002 and from 1 October to 3rd October 2003 whereas in Balochistan. Severity of disease in a field was recorded as percentage f tissue area infected out of total leaf area. The points were five (3) pates apart. For calculating actual incidence of disease in a field. More locations were visited in districts with intense cropping. Nasirabad disSs ict was examined from 2 September to 3 September 2002 and from 3 to S October 2003 In zone 4 the Indus delts. Actual incidence of disease in a field was recorded as percentage of infected plants out of total plants examined. Actual Xncide %age Number of bacterial blight Infected plants X 100 Total number of plants examined From each field upper 3 leaves of each plant were collected. t revalenee %age = Loc showing bacterial blight disease X 100 Total LocatIons visited Visual incidence of disease was recorded in terms of percentage of plants infected in a field on visual basis (IRR1. rice fields of one acre area were observed for the presence or absence of bacterial blight disease. At each point four (4) plants were examined for disease symptoms (figure S). These fnrrn composite and a representative sample was taken for isolation.
IDENTIFiCATION AND PURIFICATION OF X-INTHOMONAS ORYZAE PY. CONFIRMATION OF XANTHOMONAS ORYZAE PV. n (7) and n (9) = Number of leaves showing severity score of 1. 5 if infected area was 1325%. Finally they ware rinsed with sterilized deionized water and placed on Peptone Sucrose Agar medium. the foliar samples of plants that possessed disease symptoms were cut in to small pieces. n (3). en Total nunther of leaves scored ISOLATION OF BACTERIA FROM FOLIAR SAMPLES OF RICE From each field upper 3 leaves of the 20 plants examined were collected. Yeast Dextrose Calcium Carbonate (YDC) Agar and Wakimoto medium (appendix-I) in Petri plates at 29°C in an incubator (Wilson et o/. ORFZAE CULTURES The different types of bacterial growth obtained in th plates were accorded and those having morphological characteristics of Xanthonwna. (IRRI. Seeds of Super basniati and lltRl-6 were germinated for 41 hours in Petri dishes.score was considered 1 lithe lesion ares was 1-5 %. After 43 hours. surfaces sterilized and were plated on YDC Agar plates. Plants that were perfectly healthy and growing vigorously were selected for pathogenicity tests and were inoculated 14 days after sowing. l 967).s o pv. 3 score if area was 6-12%. After recording the observations and lesion lengths.. For calculation of disease amount in an area following formula (IRRI. These fbnned a composite nd a representative sample was taken for isolation. 3.5 and maximum score was 9 if 51. Disease n(1) -n(3)+n(5)+n(7)+n(9 Where: n (1). Then surfaces of the pieces were sterilized by dipping first in 1% Clorox for 1 minute and then in 10% ethanol for 1 minute. While the plants were still moist they were kept in growth chamber in which the temperature was controlled at spproximatcly 2l-23 and relative husnidity at 70 80%.4 days plates were . n (5).100% area of the leaf was having iesion. In order to confirm Xirirthoraanas arjzae pm o isolates by fulfillment of Koch s postulate. 7 if lesion area was 26. Light was supplied during daylight hours by artificial lights without affecting the temperature. Control plants were inoculated sinsilarly with sterilized distilled water. Nutrient Agar. To prepare the inoculusns. 1996). seedlings were transferred to pots.. the bacteria were inoculated unto two commercial rice varieties Super baamnati and IRRI-6. The tips (about I to 2 cm) of young leaves were clipped with scissors dippcd in inoculuins. Plates were observed daily for bacterial growth. Each leaf was cut into small pieces from advancing portion of the lesions. 1980). 1996) was applied. oryzas were picked with sterilized loop and purified cultures were obtained by streaking on fresh YDC medium in plates. After that. plants wore transferred to glass house at 25-27°C for development of symptoms (Sohaad. The whole experiment was conducted under controlled conditions. cells from cultures were grown in nutrient broth and were adjusted to give concentrations of approximately CFU/ml. After 3 . ORFZAE ISOLATES BY FULFILMENT OF KOCH S POSTUJ. The symptoms were keenly observed to be that of bacterial leaf blight or not.5.7 and 9.
lOg non fat. Then 3ml of sterilized milk was added irs Petri plates of nutrient agar (without glucose) having bacterial culture and made suspension by scrapping with a blue loop. ORYZAE From the sb mentioned experiment aggressiveness of X. After that it was atstoclaved at 115°C and stored in refrigerator at 4°C. For this purpose 125 x 15mm screw capped tubes were half filled with silica gel. 1999). PRESERVATION OF XANTROMONAS ORYZAE PV. (1) PRESERVATION IN SiLICA GEL Silica gel was used for preservation of Xanthoosonas oayzae pv. dry milk plus l5nsl glycerol was added to l0Oml warm water. 5. (iii) PRESERVATiON TN WATER . lips of the tithes were flamed to sterilize and granules were sprinkled on YDC medium plates and Were intubated at 29°C. The more severe rtaotio of varieties the more aggressive the strain was considered to be.. oryzae isolates thllowing the method of Sehaad (1930).1cm to 7. Then reaction rating wss done according to lesion length as follow. While using it was placed in ice bath. these producing moderetely susceptible reaction were grouped as moderately aggressive strains. Then 0. asyzree cultures. Shake in vortex mixer and placed in ice bath. garden soil wes taken. 3.1cm to 50cm as tooderately resistant reaction. EVALUATION OF AGGRESSIVENESS OF LOCAL ISOLATES OF XANTHOMONAS ORYZAB PV. oryzae pv. ORYZAE CULTURES The bacterial isolates which proved to beXartthomonas orynse pv. 1987). and those inducing susceptible reactions were categorized as highly aggressive strains. those inducing resistant reaction were categorized as non aggressive strains. To revive cultures. oryrae isolstes was also evaluated.observed f bacterial growth and the colonies were compared with mother cultures for the confirmation of Koch a postulates. few crumbs of soil were sprinkled with a sterile loop on YDC medium plates and were incubated at 29°C ( and Stead. Thou stored in refrigerator. those inducing moderately resistant reaction were classed as slightly aggressive. The suspension was stirred and filtered through cheese cloth. lml of bacterial suspension was distributed in screw capped tube (3 tubes/culture) having silica gel. screw caps were removed. (ii) PRESERVATION IN SOiL For preservation of Xwsthom ones orysae pv.grown nutrient-broth culture suspension was poured on to about 3g of the sterilized soil in small sterilized plastic tubes. oryzae by fulfilling Koch s postulates were preserved for further studies using following three methods. By applying this scale to the aggressiveness of the X oryzae pv.Sml of weil . For estimation of severity of reaction lesion lengths on the foliar samples of plants that possessed disease symptoms were measured. Cultures were stored at 4°C. <3 cm as resistant reaction.0cm as moderately susceptible reaction and 7 as susceptible reaction (Lee at a!. oryaae strains. passed through a 2mm sieve and autoelaved at 121°C on 3 successive days. To check tecovery of bacteria. After that the tubes were dried and heat sterilize at 180°C for one and a half hour. The aggressiveness of bacterial isolates was estimated from the severity of the reactions of the two varieties. Tubes were kept its plastic bags and were stored in refrigerator. It was air-dried.
An . HYPERSENSITIVITY TESTS OF LOCAL ISOLATES OF XANTHO2WONAS ORYZAE PY. 1940). (lii) STARCH HYDROLYSIS TEST Powdered nutrient agar (28 g) was dissolved in water by progressive heating. 1967). The specimen was again tinned with tap water sod counter-stsir. 1982). 1974) (ii) I OTASSIUM HYDROXIDE (KOIl) TEST Gram staining results were confirmed by 3% aqueous potassium hydroxide (KOH) solution test (Suslow at a!.ed with safranin for approximately 10 seconds. dried and observed under microscope at lOX and 40X magnifications. To check survival of bacterial cells. Plants were incubated at low temperature (18°C) for two days before inoculation and at 30-32°C after inoculation.black color of crystal violet dye (Blair at al. rinsed in tap water and decolorized with PS% ethanol until colorless run off (approximately 30 sac). Collapse of the tissue after 24 hours was considered as positive otherwise it was taken as negative (K and Goodman.5 ml of suspensions were poured on YDC media plates and spread with sterile glans spreader and were incubated at 29°C. The specimen was treated with 0. Plants were then carefully observed for their responses. oryrae isolates were performed on tobacco.5% aqueous crystal violet for 30 seconds and . bacteria were aseptically removed from Petri plate cultures With a toothpick. The specimen was then flooded with Iodine for one minute. For this purpose approximately i CFU/ ml water suspensions of freshly cultured bacteria were injected onto the intercellular apace of leaves of the three plants with hypoderrnai syringes. o isolates were cerstriflsged arid washed twice in sterile distilled water. BIOCHEMICAL CHARACTERIZATION OF XANTWOMONAS ORYZAE 1W. oryzas isolates were characterized according to their reactions to certain hiochencicul tests. Gram negative bacteria stain reddish pink whereas Gram positive bacteria retain purple to blue. Bacterial cells were resuspended after final centrifugation in sterile distilled water at a population of about l0 l0 cells per oil in sterilized plastic tubes.Well grown nutrient broth suspensions of Xanthomonas osyzae pv. Following are the protocols of the tests performed. tomato and cow pea plants. Where as Gram positive bacteria disperse in the drop and do not exhibit this reaction (Ryu. ORYZAE ISOLATES Xanthomoeas otyzse pv. 1987). placed on a glass slide in a drop of 3% KCH solution and stisted for 10 seconds using a quick circular motion of the hand.aflerwards washed with running tape water for one minute. Tubes were then kept at 4°C in refrigerator (Lelliott and Stead. ORYZAE Hypersensitivity tests of Xanthomonas oryzaa pv. The specimen was eventually washed with water. (I) GRAM STAINING A thinly spread air-dried bacterial film was fixed on a clean glass slide by a light flame. For this test. If the bacteria are gram negative. tubes were shsk and 0. Starch (2 g) was then diseolved in 10 ml distilled water separately and added to the molten agar with constant stirring. the suspension becomes viscous and forms thread-like slime when picked up with the toothpick.
tubes were placed at 4°C for 30 minutes in ice water bath prior to recording the results. (vi) ACID FORMATION FROM GLUCOSE. yeast extract (I g). The case in which gelatin was hydrolyzed. was dispensed in test tubes and autoclavsd at 121°C for 15 minutes. (V) GELATIN LIQUIFICATION TEST For Gelatin Liquificatioc test.aliquot of 100 ml of this basal medium was dispensed in conical flasks and sterilized at 115°C for 10 minutes. NaCi (5 gi. (0.2 g). CaCl (0.5% alcohol solution 0. l ANI) LACTOSE For this test 10 ml of medium containing NH.5 g). fructose and gatacrose was prepared. 14 and 21 days. 1980) (Vtf) ACID FORMATION FROM RIBOSE. Development of milky white precipitate around the colonies indicated positive reaction otherwise it was considered as negative (Sierra.5 g). After 3. four and six days.4 and then autoclaved at 121°C for 15 minutes.5 g). A 10% (w/v) aqueoua aclution of ribose. A 10% (wA ) aqueous solution of glucose. and 120g gelatin in distilled water making final volume of 36 IL. 1974). water (1 L). (lv) TWEEN 80 HYDROLYSIS TEST The basal medium used in this test was prepared by adding peptone (10 g). 1957). maltose and lactose was prepa:ed filter-sterilized through millipore injection filter and added to the molten base o . otherwise reaction was considered to be negative (Schaad. the medium flow readily as tube was gently tipped. 1974). agar (12 g) water (1 L). incubated at 27°C and checked for acid production after two.7F1 (0.2-7.7.2 g).1 g). M (0. Each isolate was transferred aseptically into a tube. the medium was unable to flow (Cowan.7 zn).5 g). K (0. inoculated by streaking and incubated at 27°C for seven days. agar (15 g) in distilled water (1 L). Tweesi 80 was added to the base to give a final concentration of 1%.7 ml). was dispensed in test tubes and autociaved at 121°C for 15 minutes. AND GALACTOSE For this assay 10 ml of medium containing NH (0. 5g peptone. The medium was then poured in Petri plates into which each isolate was transferred aseptically and incubated at 27°C for seven days. S ml of prepared medium was poured in each test tube. Media in tubes were stab inoculated with cells from 24 hour agar slants and were incubated at 27°C. While in case of unhydrolysed gelatin. bromocresoi purple (1. brornocreaci purple (1. FRUCTOSE.5% alcohol solution 0. NaCI (5 g). Production of yellow color indicated production of acid. After scraping the superfluous growth. yeast extract (1 g). Hydrolysis of starch was indicated by the presence of clear zones in the black-stained medium around or under the colonies (Cowan. MgSOe. NaCI (5 g). Tubes containing media wets plugged and sterilized for 1245 minutes at 121°C and cool until inoculated. Ki}1P0 (0. The medium was dispensed in plates. filter-sterilized through nsillipore injection filter antI added to the molten basal medium to give a final concentration of 1%. agar (12 g). fresh gelatin medium was prepared by dissolving 3g beef extract. the plate was flooded with Lugol s iodine (see appendix-I). The pH of medium was adjusted to 7.
MaCf (5 g). agar (0. 1980) (viii) OXIDASE TEST For oxidase teSt. broken aseptically and its yolk was separated into a sterile graduated cylinder. The yolk was diluted to 40% v/v with sterile water. (ix) LECITifiNASE ACTIV3TY TEST In order to check lecithinase activity for X. incubated at 27°C nd checked for acid production after two. first of all an egg yolk emulsion was prepared. supplemented with 1% glucose for 24 hours. washed well in soap solution. A loopful of inoculuns was rubbed o filter paper impregnated with 1% (w/v) aqueous tetra-rnethyl-p-phenylenediamine dihydrochloride solution. the egg was flamed. Two test tubes were inoculated for each isolate to be tested. were used as inocula. bright greenish-blue precipitate in case of positive test (MC Clung end Toabe. A platinum loop was used since traces of iron can catalyze the oxidation of th phenylene-diaznidine compound (Keane et of. An aliquot of 10 ml of the egg yolk was incorporated into 300 ml molten nutrient agar (co to 55CC) prior to pouting into the plates.3 g). (X) ANAEROBIC GROWTh TEST For anaerobic test. rinsed with water and surface sterilized with 70% ethanol for 5 minutes. Five milliliter of this basal medium was aliquoted into test tubes (3 cur diameter) and sterilized at 121°C for 20 minutes. Art aliquot (0.5 ml) of sterile aqueous glucose suspension (10%) was aseptically added to each tube containing the basal medium.give a final contentration of 1%. oiyrne pv. Moreover the plates were flooded with saturated. oryzae isolates. bacterial cultures grown on nutrient agar. 1970). bromothymol bluel% aqueous solution was prepared icr one liter distilled water and p1 was adjusted to 7. Each isolate was transferred aseptically into the tube.copper sulphate solution and observed for an insoluble. a basal medium consisting of peptone (2g). 1947). Production of yellow color indicates positive test otherwise it is considered to be negative (Sebsad. The strain was oxidase positive ifs purple colour developed within 10 seconds and delayed positive within 10-60 seconds but negative itt the absence of coloration or colour development after 60 seconds. The medium was inoculated with bacteria and incubated for 3 days at 27°C. Production of turbid zone of free fats around the colonies was considered as positive teSt otherwise it was taken as negative. One tube per isolate was over-lain with a Layer (5 torn) of aterlt . four and six days.3 g). KH (0. For this purpose a fresh egg was taken.1. Then.
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