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2004 Identification of bacterial pathogens causing panicle blight of rice in Louisiana Xianglong Yuan Louisiana State University and Agricultural and Mechanical College, [email protected]
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Recommended Citation Yuan, Xianglong, "Identification of bacterial pathogens causing panicle blight of rice in Louisiana" (2004). LSU Master's Theses. 2883. https://digitalcommons.lsu.edu/gradschool_theses/2883
This Thesis is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Master's Theses by an authorized graduate school editor of LSU Digital Commons. For more information, please contact [email protected]. IDENTIFICATION OF BACTERIAL PATHOGENS CAUSING PANICLE BLIGHT OF RICE IN LOUISIANA
A Thesis Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College In partial fulfillment of the Requirements for the degree of Master of Science In The Department of Plant Pathology and Crop Physiology
By
Xianglong Yuan B.S., University of Liaoning, 1985 M.S., Shenyang Agriculture University, 1990 May 2004
ACKNOWLEDGMENTS
I wish to express my sincerest gratitude to Dr. M. C. Rush, my advisor, for giving me this opportunity to pursue my graduate studies in the Department of Plant Pathology and Crop Physiology at Louisiana State University. It was Dr. Rush that led me to enter the world of plant pathology, widening my field of vision in academia. I sincerely appreciate his guidance, advice, understanding, and patience throughout my time of study here, particularly in the research work and writing. His working attitude will necessarily be a beacon lighting my future voyage.
I greatly appreciate Dr. A. K. M. Shahjahan’s help throughout my research efforts. His generous, patient guidance in my laboratory research, and his many excellent suggestions made during the past 2.5 years have helped my research efforts greatly.
Special thank go to my committee members, Dr. D. E. Groth, Dr. S. D. Linscombe, and Dr. J. P. Jones for their suggestions on conducting my research and writing my thesis. It was their influence that greatly inspired me in developing my thesis.
I acknowledge all of Chinese students that helped me at this university. Their great and generous support helped me to overcome many difficulties in my life and during my studies in Baton Rouge, LA.
The most sincere appreciation belongs to my mom and dad. Their encouragement, love and care were the main source of power that prompted me to accomplish this thesis. I also express thankfulness to my other family members for their generous support.
ii TABLE OF CONTENTS
ACKNOWLEDGEMENTS ...... ii ABSTRACT...... v CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW ...... 1 1.1 Disease Distribution and Economic Importance...... 1 1.2 Symptoms and Epidemiology...... 3 1.2.1 Symptoms ...... 3 1.2.2 Epidemiology...... 5 1.3 Detection and Identification Methods for Pathogenic Bacteria on Rice ...... 7 1.4 Classification, Nomenclature, and Pathological Characteristics of Pseudomonas and Related Genera ...... 8 1.4.1 Classification and Nomenclature ...... 8 1.4.2 Characteristics of Burkholderia spp...... 9 1.4.2.1 B. glumae ...... 9 1.4.2.2 B. Plantarii...... 10 1.4.2.3 The B. gladioli and B. cepacia Complex ...... 10 1.4.2.4 P. fuscovaginae, P. syringae, Acidovorax avenae ( P. avenae) ...... 11 1.5 Virulence...... 12 1.6 Disease Control...... 13 1.6.1 Chemical Control...... 13 1.6.2 Biological Control...... 13 1.6.3 Developing Resistant Cultivars and Lines Using Genetics and Biotechnology Techniques...... 13
CHAPTER 2. CLASSIFICATION, IDENTIFICATION, AND PATHOGENICITY OF BACTERIA ASSOCIATED WITH PANICLE BLIGHT IN RICE ...... 15 2.1 Materials and Methods...... 15 2.1.1 Purification of Isolates ...... 15 2.1.2 Identification of Isolates Using the BiologTM GN2 Microplate System ...... 16 2.1.3 Plant Materials ...... 17 2.1.4 Pathogenicity Tests……………………………...... 17 2.1.5 Functional Classification of the Identified Isolates ...... 21 2.2 Results and Discussion ...... 22 2.2.1 Purification and Identification of Isolates with the BiologTM GN2 System ...... 22 2.2.2 Pathogenicity Tests and Symptoms Produced ...... 38 2.2.3 Colony and Culture Characteristics of the Bacterial Pathogens ...... 47 2.2.4 Clustering of Bacterial Strains Isolated from Rice Showing Panicle Blight into Functional Groups ...... 50
iii 2.3 Summary and Conclusions ...... 56
LITERATURE CITED ...... 61 APPENDIX: BIBLIOGRAPHY OF RELATED LITERATURE...... 80 VITA...... 97
iv ABSTRACT
Four hundred and two bacterial isolates were isolated on the semi-specific S-PG medium from diseased rice tissues showing symptoms of panicle blighting. These isolates were purified using serial dilution in sterile water and replating on S-PG medium. A total of 420 single isolates were obtained. These isolates were subjected to pathogenicity tests on rice (Oryza sativa L. cv. Cypress). Based on these tests, 339 isolates were used in BiologTM tests and identified to the species level. Bacterial strains from 39 species in 16 genera were identified, including 52 isolates representing 15 Pseudomonas species and 261 isolates representing six Burkholderia species. The remaining 26 isolates included 14 other genera. Of 261 Burkholderia strains, 103 isolates were B. gladioli, 68 isolates were B. glumae, 60 isolates were B. multivorans, 25 isolates were B. plantarii, three isolates were B.cocovenenans and three isolates were B. vietnamiensis. The pathogenicity tests revealed that 69% or 234/339 isolates caused seedling infection, sheath rot, and/or panicle blighting. Most of the pathogenic strains were in the genera Burkholderia and Pseudomonas. The four most common species, B. glumae, B. gladioli, B. multivorans, and B. plantarii, comprised 90% of all of the pathogenic bacteria, suggesting that a complex of Burkholderia species were causing the panicle blight/sheath rot syndrome recently found in Louisiana. Five Pseudomonas species, with total of 16 isolates, were found to be associate with this disease, including two isolates of P. syringae pv zizanize, three isolates of P. fluorescens, three isolates of P. pyrrocinia, one isolate of P. spinosa and seven isolates of P. tolaasii. The symptoms of the disease on rice were only produced by the indicated bacterial strains. Symptoms included brown margined flag leaf sheath lesions, grain rot, sterile florets, grain discoloration, and leaf and sheath rot on inoculated plants. It was impossible to distinguish among Burkholderia species based on symptoms and colony morphology. Pathogenic Burkholderia strains produced a yellow pigment in King’s B medium. Avirulent strains did not produce this pigment. The isolates from rice were grouped by species and pathogenicity. It appeared that B. glumae and B. gladioli were the most common pathogenic species.
v CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW
1.1 Disease Distribution and Economic Importance Panicle blighting has been an important sporadic problem in the southern rice production area of the United States for many years. A somewhat similar disease called “ear blight”, pecky rice, or grain discoloration has been attributed to fungal causal agents (Lee, 1992a; Lee, 1992b). This problem is characterized by discoloration of the grain and panicle branches, usually with distinct lesions, and many fungi have been described as causing this disease (Atkins, 1974; Ou, 1985; Lee, 1992b). A symptom commonly called “panicle blight” has been observed for many years in the southern rice producing areas of the United States. It was considered to be a disorder of undetermined cause as no pathogen was isolated from diseased tissues (LSU Agricultural Center, 1987; LSU Agricultural Center, 1999; Groth, et al., 1991). The disease/disorder was characterized as having panicles with brown or straw-colored discoloration of florets (Groth et al., 1991). Panicle branches remained green, without lesions or discoloration, and affected florets stopped developing or aborted. Affected panicles had few to all of the florets diseased. When the disease was severe the panicle remained upright as the grain did not fill. In 1996 a bacterial pathogen was recognized as the cause of the rice panicle blight syndrome (Rush, 1998; Shahjahan, 1998, 2000a, 2000b, and 2000c). Rush and Shahjahan revealed that Burkholderia glumae (formerly Pseudomonas glumae) was the agent causing the bacterial panicle blight (BPB) disease in Louisiana and adjacent states. Surveying showed that the BPB occurred in 60 percent of Louisiana fields, suggesting that the bacterial pathogen was prevalent throughout the rice production areas of the state (Shahjahan, 2000b). The world wide distribution of this disease has been become clear as recent survey reports became available. Studies of the epidemiology, etiology and control of this disease have attracted the attention of rice plant pathologists from several countries. According to S.H. Ou (Ou, 1985) bacterial sheath rot and grain blighting on rice was first reported in Hungary (Klement, 1955). The disease was described as caused by Pseudomonas oryzicola (later shown by Visnyovszky et al. (1971) to be a synonym of
1 Pseudomonas syringae pv. syringae (Ou, 1985). According to Ou (1985), research on bacterial grain rot was initiated by Goto in Japan (Goto and Ohata, 1956; Goto et al.,1987). Grain rot in rice was reported to be caused by several bacteria, including Pseudomonas glumae (Burkholderia glumae), but only P. glumae caused seedling blight on inoculated plants (Goto et al., 1987). According to Hashioka (1969); Kurita and Tabei, 1967 named the pathogen of grain rot Pseudomonas glumae Kurita et Tabei. The temperature optimum for growth of P. glumae was reported as 30-35C with a range of 11-40C and a thermal death point at 70C (Kurita et al., 1964). Tanii, et al. (1974) isolated several bacteria from rice grains showing grain rot. They identified three Pseudomonas species, but not P. glumae. P. glumae was isolated from plants in nursery flats with seedlings showing bacterial seedling rot symptoms (Uematsu, et al., 1976). Since the 1980’s, the disease has been reported from several different countries in the world, including Korea (Jeong et al., 2003), Taiwan (Chien and Chang, 1987), Latin America (Zeigler et al., 1987; Zeigler and Alvarez, 1989a; 1989b; 1989c; 1990 and Zeigler, 1990), Vietnam (Trung et al., 1993), Philippines (Cottyn et al., 1996a; 1996b) and the Gulf of Mexico rice production area in the U.S. (Rush, et al.,1998; Shahjahan et al., 1998; 2000b; 2000c). It is reasonable to believe that this disease frequently occurs in rice around the world, particularly in tropical countries. It is not surprising as the pathogen is readily seedborne. Rice is considered to be one of the most important food crops in the world (Lu and Chang, 1980). It is clear that wide-spread development of a major rice disease would be disadvantageous to the world’s economy. The direct agronomic consequences of such an event would be a severe decrease in crop yield and stability of production. BPB has the potential to reduce the yield of rice as much as 75% in severely affected regions due to reduction in grain weight, floret sterility, inhibition of seed germination, reduction of stands, as well as the year-to-year transmission because of the seedborne nature of the pathogen (Trung, et al., 1993).
2 1.2 Symptoms and Epidemiology 1.2.1 Symptoms The reported symptoms for this disease syndrome were observed in Louisiana on the leaf sheath of seedlings, flag leaf sheath, and panicle florets. The disease was described as sheath rotting (brown, necrotic lesion with a distinct margin); leaf stripe; flag sheath browning (Figure 1); grain rot (Figure 2); spikelet discoloration (grayish or straw-colored, with bottom half of the floret darkening); sterile florets and/or seedling blighting ( Shahjahan, 2000b). Several bacterial species have been proposed as causing similar symptoms. The suspected pathogens are mainly distributed in two taxonomically and physiologically close genera, Pseudomonas and Burkholderia. B. glumae causes rice grain rot and seedling rot (Uematsu et al., 1976), sheath rot complex, and grain/sheath rot (Cottyn et al., 1996a, 1996b, and 2001). The bacterial stripe disease, with grain discoloration, was reported as caused by Pseudomonas avenae (Kadota and Ohuchi, 1983). Sheath rot and grain discoloration in Latin America was believed to be caused by Pseudomonas fuscovaginae and Pseudomonas glumae (Zeigler and Alvarez, 1987; Zeigler and Alvarez, 1989b). However, the different pathogens share similar symptoms among them. For instance, P. syringae, P. fluorescens and B. glumae caused similar early symptoms (Goto et al., 1987). Zeigler reported the symptoms produced by Pseudomonas fuscovaginae as extensive water-soaking and necrosis with poorly defined margins and glume discoloration before panicles emerge from boots. Infected grain varied from completely discolored and sterile to nearly symptomless with only small brown spots (Zeigler and Alvarez, 1987; Zeigler et al., 1987; Zeigler and Alvarez, 1989a; Zeigler and Alvarez, 1989c). Symptoms typically caused by B. glumae in Louisiana were panicle blighting with floret discoloration (with a gray-brown color), usually on the lower half of the developing grain, with a clear deep brown border followed by sterility or partial filling of the florets causing the panicles to stand erect (Shahjahan et al., 1998; Shahjahan et al., 2000b). It is clear that accurate identification of the pathogens and epidemiological surveys are essential before proposing practical control schemes.
3
Figure 1.1. Flag leaf sheath rot, caused by Burkholderia glumae on inoculated Cypress rice.
4
Figure 1.2. Panicle blighting and grain rot, caused by Burkholderia glumae, on inoculated Cypress rice.
1.2.2 Epidemiology Bacterial pathogens of plants exist ubiquitously in the plant’s ecosystems and are usually found in the air, soil, and water. Soil distributions of these pathogens are affected by soil type, pH value, plants cultivated, and weather conditions. It has been established that pathogenic bacteria exist on the phylloplanes of rice plants during the growing season (Matsuda and Sato, 1987; Hikichi, 1993a; Hikichi, 1993b; Hikichi et al., 1993; Otofuji et al., 1988; Tsushima et al., 1996), on or in rice seeds stored at room temperature during the winter (Tsushima et al.1987; Tsushimi et al., 1989), on weeds in the field and
5 in rice tissues from the previous crop buried in soil (Sogou and Tsuzaki, 1983). The disease can develop from inoculum in infected seeds from the previous year, in soil, and on weeds in the field. In the process of infection, host susceptibility, inoculum density, and climatic factors play the key roles (Tsushima et al., 1985; Tsushima et al., 1987; Tsushima and Naito, 1991; Tsushima et al., 1995a; Tsushima et al., 1996; Tsushima, 1996). This disease tends to break out under conditions of unusually high temperatures, especially at night, and frequent rains (Tushima et al., 1985; Mew, 1992; Zeigler and Alvarez, 1990). In 1995 and 1998 there were severe incidences of rice BPB in Louisiana as well as the other Southern rice production areas. Yield losses as high as 40% were observed in some fields (Shahjahan, 2000b). Record high temperatures were recorded during these seasons, with high temperatures extending into the night. High humidity levels at the flowering stages were reported to be particularly conductive to spikelet infections (Tsushima et al., 1995b). The most susceptible period for floret infections appears to be during panicle emergence and flowering. Inoculation during flowering gives the highest rates of floret infection and production of diseased spikelets (Shahjahan et al., 1998). In these tests using B. glumae, a high level of floret infection continued for up to 3 days after flowering in inoculation experiments. The infection rate of the pathogen also depends on inoculum density. Pot experiments conducted by spraying emerging panicles revealed that infection increased proportionally as the lognormal volumes of inoculum density (Tsushima et al., 1985). Based on their experiments, 102 to 104 cfu was proposed as the minimum inoculum density when spraying in the field and not less than1 cfu/ml was an appropriate dose for the injection method (Hikichi, et al. 1994). Pathogen cells present on leaf sheaths play an essential role in primary infection. Infection on the leaf sheath provides the primary source of inoculum to the emerging panicle (Tsushima et al., 1991 and Tsushima et al., 1996). We have also observed that that following injection of the pathogen into boots the first visible diseased tissue was always on the flag leaf sheath followed by panicle infection. When rice plants are developing from apparently healthy seeds, the uninfected flag leaf sheath forecasts that the panicle should be healthy. Therefore, the frequency and severity of infection of the
6 flag leaf sheath is an important indicator of the eventual infection on the panicle because the sheath is spatially close to panicles and the primary time of infection was at heading time (Tsushima, 1996; Tsushima et al., 1996). The primary infection site by B. glumae is apparently through the plumules (Hikichi, 1993a; Hikichi, 1995b). The bacterium enters the lemma and paleae through stomata, multiplies in intercellular spaces of the parenchyma (Tabei et al, 1989), and moves towards the surrounding healthy cells and tissues. The bacteria were detected in the epidermis, parenchyma, and sclerenchyma of glumes of naturally infected rice seeds using antiserum (Hikichi, 1993b). The long-distance movement of bacteria was accomplished via vascular systems. Bacteria were observed, by the author (X. Yuan), flooding out of vascular systems of severely infected rice by tissue sectioning techniques.
1.3 Detection and Identification Methods for Pathogenic Bacteria on Rice After evaluating the efficacy of the various methods, it appeared important to select an appropriate combination of methods to recognize the pathogens. A good identification scheme depends not only on developing a satisfactory resolution level of methods, but also on the group of bacteria studied (Welch, 1991). Apparently, it is difficult to differentiate pathogens that are closely related physiologically and taxonomically by the symptoms they produce and by their growth on selective media. A semi-selective medium for B. glumae was developed by Tsuschima et al., 1986; however, other Burkholderia species will also grow on the SP-G medium. Traditional biochemical and serological methods still have powerful advantages. Cottyn et al., 1996a and 1996b detected 204 pathogens strains distributed among seven species by using a comprehensive scheme including antiserum, API 20NE, BiologTM, and cellular fatty acid methyl ester-fingerprinting. The best differentiation was given by the BiologTM system (Cottyn et al., 1996b). Although immunological methods possess quiet high resolution, specific antibodies to some antigens are not readily obtained and this may limit the wide application of these methods. Recently, PCR-based DNA-typing methods based on both conservation and variance of rDNA genes among species and pathovars, and other molecular based techniques, have become popular due to the rapidity, accuracy, and relative simplicity of operation. These methods will undoubtedly become powerful tools
7 in the field of classification and identification of microorganisms (Furuya et al., 2002; Karpati and Jonasson, 1996; Mizuno et al., 1995; O’Callaghan et al., 1994; Pelt et al., 1999; Reiter et al., 2000; Tyler et al., 1995; Ura et al., 1998; Whitby et al., 2000a; Whitby et al., 2000b.
1.4 Classification, Nomenclature and Pathological Characteristics of Pseudomonas and Related Genera
1.4.1 Classification and Nomenclature It has long been known that some species in the genus Pseudomonas were plant pathogens. According to Burkholder (1949), as early as 1921 Pseudomonas gladioli was identified as the causal agent of flower rot of gladiolus. Burkholder described a new bacterial pathogen causing onion sour skin and rots and proposed the bacterium as Pseudomonas cepacia n. sp. (Burkholder, 1949)., However it was not recognized that some Pseudomonas spp. could serve as severe human pathogens until the 1960’s. Recently, it has been confirmed that B. cepacia and B. gladioli can produce human cystic fibrosis disease (CF) and pulmonary disease (Baxter et al., 1997; Parke and Gurian- Sherman, 1998; Vandamme, 1996, Vandamme et al., 1997, and Vandamme et al., 2000). The genus Burkholderia was not established until 1992 when Yabuuchi proposed to transfer seven species previously in the Pseudomonas homology group II to the new genus Burkholderia (Yabuuchi et al., 1992). P. plantarii and P. glumae were transferred to this genus in 1994 (Urakami et al., 1994). Yabuuchi et al. reclassified two Burkholderia species, B. solanacearum and B. pickettii, and Alcaligenes eutrophus, into the novel genus Ralstonia (Yabuuchi et al., 1995). Until 2002, there were 28 species in the genus Burkholderia (Table 1.1). Many of the species were believed to be plant pathogens causing rice, tobacco, maize and other crop diseases. The established rice pathogens included P. avenae (Goto and Ohata, 1956), P. fuscovaginae (Zeigler, 1987, 1989, 1990; Rott, 1989 and 1991; Cottyn, 1996 and 2001), P. syringae, B. glumae (Goto and Ohata, 1956; Goto,1965 and Goto and Ohata,1987; Hikichi et al., 1993d, Hikichi et al.,1994, and HIkichi et al.,1995a; Tsushima et al., 1985, Tsushima et al.,1986, and Tsushima, 1991; Cottyn et al., 1996a, 1996b and 2001), B. plantarii (Azegami et al.
8 1985, Azegami et al.,1987 and Azegami et al.1988), B. gladioli (Cheng, et al., 2001), and B. vietnamiensis (Trung et al., 1993).
Table 1.1. Bacterial species described in the genus Burkholderia.*
B. andropogonis B. cepacia genomovar I B. anthina B. multivorans (formerly genomovar II) B. ambifaria B. cepacia genomovar III B. caledonica B stabilis (formerly genomovar IV) B. caribensis B. vietnamiensis (formerly genomovar V) B. caryophylli B. cepacia genomovar VI B. cocovenenans B. plantarii B. fungorum B. pseudomallei B. gladioli B. phenazinium B. glathei B. pyrrocinia B. graminis B. sacchari B. glumae B. thailandensis B. kururiensis B. ubonensis B. mallei B. vandii * Coenye et al., 2001; Estrada-de los Santos et al., 2001; Gillis et al., 1995; Vandamme, 1996; Vandamme et al.,1997; Vandamme et al., 2000; Yabuuchi et al., 1992.
1.4.2 Characteristics of Burkholderia spp. 1.4.2.1 B. glumae B. glumae was first recorded as a rice pathogen in the genus Pseudomonas (Goto and Ohata, 1956), and is considered the major bacterial agent causing seedling and grain rot in Japan. The typical symptoms were described as sheath brown rot (Cottyn et al., 1996a and Cottyn et al., 1996b), grain rot and/or discoloration (Cottyn et al., 1996; Goto and Ohata, 1956 and Goto et al., 1987; Tsushima et al., 1986; Mew et al., 1990; and Zeigler and Alvarez,1989a.), and seedling rot (Uematsu et al., 1976a and Uematsu et al 1976a; Tsushima et al., 1986). B. glumae is a nonfluorescent bacterium producing a yellow-green, water-soluble pigment on various media. The organism is rod-shaped, with 1-3 polar flagella. The colony is grayish white or yellow due to the pigment. Arginine dehydrolase, oxidase reaction and nitrate reduction express a negative reaction. Lecithinase production and the utilization of L-arginine and inositol are positive.
9 1.4.2.2 B. plantarii The existence and pathogenicity of B. plantarii was first recognized in northern rice production areas in Japan (Azegami et al., 1987). The pathogen was found to be distributed on the weeds in fields and in seed stored at room temperature. B. plantarii is often isolated when isolating B. glumae, suggesting that they may share a similar transmission path and life cycle. They also show similar symptoms on rice (Azegami et al., 1987 and Azegami et al., 1988; Tanaka et al., 1994). However, so far this organism has only been reported from two countries. Azegami et al., (1987) reported that B. plantarii produced a reddish-brown pigment on media. 1.4.2.3 The B. gladioli and B. cepacia complex
B. gladioli and B. cepacia are organisms with wide distribution, and have been isolated from soil, water, plant roots, plant rhizospheres, and the lungs of CF patients (Holmes et al., 1998; Segonds et al., 1999; Vandamme et al., 1997). For instance, B. multivorans (B. cepacia genomovar III) was found to be a common plant-associated bacterium (Balandreau et al., 2001), and can colonize rice, corn, maize, pea, sunflower, and radish. In natural niches, B. gladioli and B. cepacia regularly coexist with each other, with B. glumae, and/or occur in the form of hybrids of B. gladioli and B. cepacia. These hybrids may have a significant pathogenic importance (Baxter et al., 1997). Cheng et al. (2001) revealed that B. gladioli was one of the causal agents of rice BPB in Louisiana. In Japan, the symptoms on rice infected with B. gladioli were reported as necrosis or chlorotic spots on the leaf (Furuya et al., 1997). In addition, necrosis with water-soaked lesions on tobacco leaves was recorded (Furuya et al., 1997), revealing that this bacterium has a wide host range. In addition to being a human pathogen, B. cepacia has been reported to promote maize growth (Bevivino et al., 1998), to enhance crop yields (Chiarini et al., 1998; Tabacchioni et al., 1993), to suppress many soilborne plant pathogens (Bevivino et al., 1998; Hebbar et al., 1998; McLoughlin et al., 1992), and to degrade diverse pesticides (Daubaras et al., 1996; McLouglin et al., 1992). B. cepacia has demonstrated an effective role in biological control of soilborne, foliar, and post-harvest plant diseases. However, it has recently been recognized that this application may pose risks to human health and
10 create environmental problems (Holmes et al., 1998). Aspects of symptomology and epidemiology on rice concerned with this organism still remained little reported. 1.4.2.4 P. fuscovaginae, P. syringae and Acidovorax avenae (P. avenae) Another widely observed pathogenic bacterium on rice is the nonfluorescent bacterium P. fuscovaginae, which produces symptoms similar to those produced by the previously mentioned bacterial pathogens on rice, and has a worldwide distribution. Ziegler and Alvarez, (1987a) reported that the bacteria P. fuscovaginae and P. syringae pv. syringae caused sheath brown rot and grain and sheath discoloration on rice in Latin America. P. fuscovaginae, P. syringae pv. syringae, A. avenae, and B. glumae were all identified from rice in Colombia and Central America (Zeigler and Alvarez, 1987b; Ziegler and Alvarez, 1989a; Ziegler and Alvarez, 1989b; Ziegler, 1990).. Of them, P. fuscovaginae and A. avenae were the most common pathogens (Zeigler, 1990). Rott et al., (1989) showed that there were intra-species differences among the native strains of P. fuscovaginae in Madagascar and reference strains by serological techniques. This suggested that epidemiology studies and surveys could be complicated by strain differences. P. syringae was first detected by Kuwata (1985) on rice in Japan where it caused the halo blight disease on rice. This new rice bacterial disease had symptoms of small brown foci surrounded by large, yellow halos on leaf blades (Kuwata, 1985). The disease on rice caused by A. avenae (formerly P. avenae) shows brown stripes on leaf blades and sheaths extending over the whole leaf blade in the severest infections (Ou, 1972; Kodota and Ohuchi, 1983; Shakya and Chung, 1983; Shakya et al., 1985; Tominaga et al., 1983). The pathogen has a wide host range, causing diseases on barley, maize, millet, oats, and rice (Kadota, 1996). The research of Cottyn et al. (1996a and 1996b) demonstrated the existence and pathogenicity of Philippines strains of P. fuscovaginae, P. syringae pv. syringae, A. avenae, and B. glumae. However, his reports simultaneously pointed out that none of these bacteria could be associated with distinct symptoms and B. glumae could not be differentiated from P. fuscovaginae, P. syringae, and A. avenae based on symptoms, suggesting that the limitations of this method indicated that a comprehensive detection scheme and more advanced techniques appeared to be necessary.
11 1.5 Virulence Sato et al. (1989) isolated two bioactive substances from a culture filtrate of B. glumae, identified as toxoflavin and fervenulin, and indicated that these phytotoxins could induce chlorotic spots on detached rice leaves. Toxoflavin, a yellow crystalline solid, first isolated in 1933 from B. cocovenenans (Buckle et al., 1990), reduces the elongation of sprouts and roots of rice seedlings (Suzuki et al., 1998; Yoneyama et al. 1998). When rice panicles were treated with the substance a brown band on the palea and lemma, a characteristic symptom of bacterial grain rot, was observed (Liyama et al., 1995). This phytotoxin was also detected in B. glumae, several strains of B. gladioli (Suzuki et al, 1998), and B. cocovenenans, not only from culture filtrates, but also from rice seedlings infected with the bacteria (Liyama et al. 1994; Liyama et al., 1995; Liyama et al., 1998). Wang compared the virulent effects of pigment producing strains and non- producing strains of B. glumae on rice seedlings and potato tuber slices. The results indicated that all of the 28 pigment producing strains used were virulent to rice seedlings, whereas all of non-producing strains were avirulent with two exceptions (Wang et al., 1991). Liyama et al.(1994 and 1995) also demonstrated that virulence to rice was closely correlated with the toxin after making comparisons between the phytotoxin producing strains and non-producing strains. By using a transposon mutagenesis technique that destroys the genes encoding toxin-biosynthesis or virulence-related pathways, transposon mutants of B. glumae were obtained. The results showed that the non-toxin producing mutants were unable to induce rice bacterial rot disease (Yoneyama et al., 1998). In addition, there is a report indicating that the virulence of B. glumae declined rapidly with continued transfers on PSA medium, suggesting that the loss of virulence occurs from subculture to a subculture (Tsushima et al., 1991). The virulence of B. plantarii was associated with production of the phytotoxin tropolone, a water-soluble substance, which is produced by some Pseudomonas and Burkholderia spp. including B. glumae. This compound is bioactive to plants, fungi, and bacteria (Lindberg et al., 1980; Lindberg,1981). This toxin is a non-benzenoid aromatic compound with a seven-member ring and gives red crystals of ferric tropolone when
12 chelated with iron. It is not clear whether the reddish-brown pigment on B. plantarii growth media (Azegami et al., 1985 is associated with tropolone production.
1.6 Disease Control 1.6.1 Chemical Control Many bactericides are able to effectively control or suppress the occurrence of seedling rot and panicle rot caused by plant pathogenic Burkholderia spp. including antibiotics, copper, and copper-containing compounds (Katsubi and Takeda, 1998; Shahjahan et al., 2000b). Oxolinic acid, a synthetic bactericide developed from quinoline derivatives, inhibits disease development by Gram negative bacteria. This compound was highly efficacious for the control of this rice disease, either as seed treatments or foliar sprays. It exhibited both preventive and curative effects (Hikichi, 1993). A study of the efficacy of oxolinic acid, with FITC-conjugated antibody and fluorescence microscopy, demonstrated that only 3% of seedlings expressed measurable symptoms after treatment, compared with 92% of seedlings from untreated seeds (Hikichi, 1995). 1.6.2 Biological Control The ability of some avirulent strains of Burkholderia to restrict the development of rice grain rot has been confirmed. Spraying rice panicles with a mixed suspension of a virulent strain of B. glumae and an avirulent strain of B. gladioli almost completely controlled the occurrence of the disease in pot and field experiments (Miyagawa and Takaya, 2000). Similar efficacy was expressed when the seedlings were sprayed with the avirulent B. gladioli strain prior to inoculation with the B. glumae strain; whereas no suppression was observed when the order of inoculation was reversed (Miyagawa, 2000a). Similar results were also observed using avirulent strains of B. glumae (Furuya, et al., 1991) and of B. plantarii (Ohno, et al., 1992), indicating that pre-treatment of seeds with avirulent strains is an effective biological control method.
1.6.3 Developing Resistant Cultivars and Lines using Genetics and Biotechnology Techniques Developing resistant cultivars and lines using genetic and biotechnological means is one of the major trends in plant disease control. Transforming rice with the oat thionin gene gave a measurable resistance against grain rot caused by B. plantarii (Iwai, et al.,
13 2002). Due to a high level of accumulation of thionin in cell walls, this transgenic rice was protected against the attack of the bacterium. The stained and marked bacteria distributed only on the surface of stomata, whereas, all of the inoculated wild-type rice Seedlings, developed from seeds germinated after treatment with bacterial suspension, were wilted and showed characteristic blight symptoms (Iwai, et al., 2002). Rush revealed that B. cepacia and B. gladioli were common inhabitants of the phylloplane of rice in Louisiana (Rush et al., 1998). Previous studies showed that B. glumae (Shahjahan, 2000a and 2000b) and B. gladioli (Cheng, et al, 2001) were the causal agents of BPB on rice in Louisiana. However, it is still unclear whether other bacterial pathogens exist that can cause this disease. In this study more than 400 bacterial isolates obtained from infected rice panicles collected from rice fields in Louisiana, were used to conduct pathogenicity tests and the cultures were grouped into functional, biochemically characteristic strains for proper identification with the BiologTM system.
14
CHAPTER 2
CLASSIFICATION, IDENTIFICATION, AND PATHOGENICITY OF BACTERIA ASSOCIATED WITH BACTERIAL PANICLE BLIGHT IN RICE
Several hundred strains of bacteria isolated from diseased tissues of rice collected in Louisiana and showing BPB symptoms were provided by M.C. Rush and A.K.M. Shahjahan for further purification, for identification to species, to conduct pathogenicity tests to determine which strains were pathogens, and to determine the virulence of pathogenic isolates. The objectives of this study were to: 1. Replate by serial dilution and streaking all of the primary cultures provided by Dr. A.K.M. Shahjahan. Obtain single bacterium cultures from the primary isolates, and preserve these cultures for further study.
2. Identify all single bacterium cultures to species using the Biolog TM GN MicroPlate System (Jones, 1993).
3. Conduct pathogenicity tests on rice seedlings and panicles in the boot by injection of bacterial suspensions of all of the identified bacterial cultures..
4. Identify bacterial isolates pathogenic on rice and quantify virulence of the pathogenic isolates.
2.1 Materials and Methods
2.1.1 Purification of Isolates Bacterial isolates isolated previously on the selective medium S-PG (Tsushima et al., 1986), from diseased grains or rice sheaths (Oryza sativa L.) in samples collected from Louisiana rice production areas, were used in this study. All of the bacterial isolates were subcultured on King’s Medium B with agar (KBA) (20g protease peptone #3, 1.5g . KH2PO4, 1.5g MgSO4 7 H2O, 15 g Bacto-agar, and 15 ml glycerol). All isolates were purified by serial dilution with sterile distilled water and plating on KBA medium. Plates
15 were incubated at 30C for 48 hr. The bacterial colonies on these plates were examined for their morphological characteristics. Single colonies, with different cultural characteristics, from each culture plate were touched with a flamed bacteriological loop and streaked on KBA medium, incubated at 30C for 48 hr, and then stored in microcentrifuge tubes at -70oC in 30% glycerol. Each isolate was given a culture number.
Figure 2.1.1. Typical colonies of Burkholderia sp. on S-PG medium.
Bacterial strains were removed from the freezer as needed for identification or pathogenicity tests, plated on KBA medium, and incubated at 30C for 48 hr. The development or failure of development of yellow pigment in the medium was noted for selected B. glumae and B. gladioli isolates for comparison of pigment production with pathogenicity of these cultures (Figures 2.25, 2.26, and 2.27).
2.1.2 Identification of Isolates Using the BiologTM GN2 Microplate System Test isolates were removed from the freezer, plated on KBA medium, and incubated at 30C for 48 hr. Bacteria were subjected to Gram staining tests (Brock et al, 1994). Gram-negative cultures were transferred onto BUG Agar (BUG Agar with 5% sheep blood), and incubated at 30C for 24hr. Bacterial cells were removed from the plates with sterile cotton-tipped swabs, suspended in a GN/GP-IF containing 18 to 20 ml of sterile “gelling” inoculating fluid (0.40% NaCl, 0.03% Pluronic F-689, 0.02% Gellan Gum). Cell densities were adjusted with a turbidimeter to 52+/-4% transmittance. The BiologTM GN2 MicroPlate test panels (Biolog, Hayward, CA) were inoculated and incubated at 30C for 24-48 hr. Results were analyzed with BiologTM GN2 database
16 version 4.20 to determine the identity of each isolate. Data on each isolate was printed out and kept in ring binders.
2.1.3 Plant Materials Ten to 15 rice seeds of the variety Cypress were planted in each 15-cm plastic pot with soil consisting of a mixture of 2:1:1 soil, sand, and peat moss, respectively. The plants were grown under natural light with a day temperature of 33C – 41C and 75% - 95% relative humidity in a greenhouse on the Louisiana State University campus in Baton Rouge, LA. Fertilizer was applied in the form of 1 tablespoon of slow release OsmocoteTM with NPK of 19-5-8 for general plant maintenance. After seedlings were established they were thinned to a population of 5-8 seedlings per plot (Figure 2.1.2).
2.1.4 Pathogenicity Tests Bacterial inoculum grown on KBA medium incubated at 30C for 48 hr were harvested with a sterile cotton swab and suspended in a vial containing 0.9ml of sterile distilled water (ca. 107-108 cfu/ml). All seedling inoculations were by injection with sterile B-D TM 1ml syringes using B-DTM 23G1 needles. Inoculation preparation and injection of bacteria into plants was conducted using sterile technique. The person inoculating plants wore rubber gloves and rubbed his hands in the gloves with 95% ethanol. Three to five Cypress rice seedlings in the same pot were inoculated on stems before the plants began tillering. Inoculum consisted of 0.5ml of each bacterial isolate per stem. The inoculated seedlings were maintained in the greenhouse. After 2 weeks the disease reaction was determined on each inoculated plant. Inoculated seedlings were rated with a four category scale. In this scale 0 = no symptoms produced after inoculation (Figure 2.1.3), + = slight browning around the injection site, ++ = a brown lesion 1-2 cm in diameter or spreading up and down the stem from the injection site with a distinct darker brown lesion, +++ = brown lesion spreading up the stem from the injection site and with the leaf blade on the inner leaf yellowing or blighted and turning necrotic (Figure 2.1.4). Plants with a “0” rating were listed as NP or non-pathogenic in the data tables.
17
Figure 2.1.2. Four-leaf-stage Cypress rice seedlings ready for inoculation in pathogenicity tests.
Figure 2.1.3. Inoculated seedling showing “0”or NP reaction.
18
Figure 2.1.4. Inoculated seedlings showing symptoms typical of a +++ rating (picture courtesy of A.K.M. Shahjahan).
Cypress plants produced as above were fertilized again at the maximum tillering stage with 1 tablespoon of OsmacoatTM, and inoculated just before panicle emergence (Figure 2.1.5) with 1.0ml of bacterial suspension injected into the boot using B-DTM 3ml syringes with B-DTM 23G1 needles. Inoculated panicles were rated 3-4 weeks after inoculation and emergence with a similar rating where 0 = no signs of disease on the panicle or florets, + = some browning of the florets with most grain filling normally; ++ = browning on most florets and some grain not filling, lesions formed on sheaths at inoculation points, +++ = panicles poorly emerging, or fully emerging and florets discolored and brown with many florets sterile and panicles tending to remain upright due to lack of grain filling ( Figure 2.1.6). An elongated lesion usually developed on the flag-leaf sheath at the inoculation point with virulent strains (Figure 2.1.7).
19
Figure 2.1.5. Plants at the boot stage ready for inoculation.
Figure 2.1.6. Typical +++ reaction for panicles inoculated with a virulent Burkholderia strain.
20
Figure 2.1.7. Flag leaf sheath rot caused by Burkholderia glumae on inoculated Cypress rice.
All final pathogenicity ratings and evaluations were made during July to October in the greenhouse with day-time temperatures ranging from 37-43C and relative humidity ranging from 75-95%. The high temperatures and humidity favored disease development. Please note that seedling reactions made in the summer of 2002 were not rated with the four reaction scale, but were assigned a not pathogenic (NP) or pathogenic (P) rating.
2.1.5 Functional Classification of the Identified Isolates The isolates of the four most common bacteria, B. glumae, B. gladioli, B. multivorans, and B. plantarii, were clustered into functional groups based on two clustering systems used independently. One was based on the four unit scale of severity and the other was a clustering system based upon the similarity index of an isolate to a typical strain in the BiologTM database. This was done to evaluate the correlation between the strains and identification accuracy. An isolate was separated into a group of high-similarity index or a group of low-similarity indexes based on the BiologTM identification index.. The threshold value of high or low similarity for B. plantarii was assigned to be 0.6. Considering larger number of isolates in B. glumae, B. multivorans
21 and B. gladioli, a weight was added to the threshold values of each of these species. The threshold value of high-similarity or low-similarity for B. multivorans and B. glumae was shifted up to 0.65 and the index was shifted to 0.7 for B. gladioli. By combining both clustering systems, 8 groups of bacteria in B. glumae, B. gladioli, B. multivorans, and B. plantarii were produced; that is, high virulence + high similarity index, high virulence + low similarity index, low virulence + high similarity index, low virulence + low similarity index, not virulent + high similarity index, not virulent + low similarity index, moderate virulence + high similarity index, and moderate virulence + low similarity index. The results of classifications were represented in Tables 2.2.7, 2.2.8, 2.2.9, and 2.2.10. The distribution characteristics of isolates of B. gladioli and B. glumae after such classifications were illustrated in Figures 2.2.24, 2.2.25, and 2.2.26.
2. 2 Results and Discussion 2.2.1 Purification and Identification of Isolates with the BiologTM GN2 System Based upon the previous isolation of bacteria using the S-PG semi-selective medium, a total of 402 bacterial isolates were isolated and further purified using serial dilution in sterile water. This procedure gave 420 bacterial isolates. All of these isolates were subjected to pathogenicity tests based on inoculating Cypress seedlings at the four- leaf stage of growth and panicles in the boot just before emergence. Of these isolates, 339 were identified to the species or pathovar level using the BiologTM GN2 system (Figures 2.2.1, 2.2.2, 2.2.3, 2.2.4, and 2.2.5). The bacteria belonged to 39 species distributed among 16 genera (Table 2.2.1). Burkholderia was the most common genus, giving 262 isolates. One hundred and three isolates were B. gladioli, 68 isolates were B. glumae, 25 isolates were B. plantarii, 60 isolates were B. multivorans, 3 isolates were B. cocovenenans, and 3 isolates were B. vietnamiensis. Fifty two isolates were placed in the genus Pseudomonas; and another 26 isolates were distributed among 14 other genera (Table 2.2.1) Pathogenicity tests on the variety Cypress confirmed that the bacteria that were pathogenic consisted of 234 of 339 strains tested or 65% of the isolates (Table 2.2.2). Most of the pathogenic strains were in the two genera Burkholderia and Pseudomonas. The four most commonly isolated species, B. glumae, B. gladioli, B. multivorans, and B.
22 plantarii, comprised 90% of the pathogenic bacterial strains, indicating that a complex of Burkholderia species may be the causal agents of the BPB and sheath rot disease recently identified in Louisiana (Table 2.2.2). This is apparently the first report of B. multivorans and P. tolaasii ( Table 2.2.3) as bacterial pathogens of rice. Also, this is the first time that B. gladioli and B. plantarii were clearly determined to be pathogens of rice in Louisiana. Among the B. glumae isolates tested, 91% were pathogenic on Cypress, suggesting that there is widespread pathogenicity among natural strains of this species (Table 2.2.2). Three isolates were tentatively identified as B. cocovenenans and three as B. vietnamiensis. If these are valid identifications, this suggests that these species only rarely infect rice in Louisiana. No isolates of P. fuscovaginae, which was previously reported as a causal agent of grain discoloration and grain rot in Latin America (Zeigler and Alvarez, 1987a and Ziegler and Alvarez, 1990) and in Africa (Rott et al., 1989 and Rott et al., 1991), was detected among bacteria isolated from diseased rice in Louisiana. It was evident that most of the Pseudomonas species, although associated with Burkholderia species in epiphytic communities on diseased rice tissues, were not the causal agents. Five of 15 Pseudomonas species showed pathogenicity on Cypress rice (Table 2.2.3). Two strains of P. syringae pv zizanize, isolated from diseased grains, showed pathogenic characteristics to both rice seedlings and panicles similar to that reported in the literature. P. syringae pv tagetis isolated from a diseased rice sheath was not pathogenic in inoculation tests. Three strains of P. pyrrocinia were rice pathogens consistent with previous reports in the literature (Table 2.2.3) (Rott et al., 1989). Seven strains of Acidovorax avenae, including two isolates of A. avenae subsp. avenae and five isolates of A. avenae subsp. cattleyae, were not pathogenic on rice seedlings or panicles although Zeigler (1990) reported A. avenae as pathogenic in Latin America. These two subspecies were non-pathogens. In addition, an isolate identified as Vibrio tubiashii by the BiologTM System, caused intermediate pathogenicity on inoculated rice seedlings in this study. Whether it is a new plant pathogenic species needs further research. For most of the bacterial strains tested, isolates were pathogenic to both seedlings and panicles (Table 2.2.2). However, there were 26 instances among Burkholderia strains where the strain was pathogenic on panicles, but not on seedlings. Only one strain was
23 pathogenic on seedlings but not on panicles. This suggests that panicles in the boot or emerging are more susceptible than 4-leaf-stage seedlings.
TM Figure 2.2.1. Biolog GN2 Profile of B. gladioli isolate 382gr-1.
Figure 2.2.2. BiologTM GN2 profile of B. multivorans isolate 99sh-2.
24
Figure 2.2.3. Biolog GN2 Profile of P. syringae pv. zizaniae isolate 319gr-4.
Figure 2.2.4. BiologTM GN2 Profile of B. plantarii isolate 260gr-3.
Figure 2.2.5. BiologTM GN2 Profile of B. glumae isolate156sh-1-b.
25 Table 2.2.1. Genera of bacteria isolated from panicle blighted rice plants collected in Louisiana. Genera isolated Number of species in each genus Burkholderia 6 Pseudomonas 15 Acidovorax 2 Agrobacterium 1 Aquaspirillum 1 Aeromonas 1 Commonas 1 Chryseobacterium 1 Flavimonas 1 Flavobacterium 1 Herbaspirillum 1 Pasteurella 1 Rhizobium 1 Sphingomonas 2 Stennotrophomonas 1 Vibrio 3
Table 2.2.2. The frequency of isolation of Burkholderia species from rice plants showing symptoms of bacterial panicle blight.* Frequency of distribution Number of isolates Species Identified of the species among of each genus Burkholderia isolates
B. glumae 68 26% B. gladioli 103 39.3% B. multivorans 60 22.9% B. plantarii 25 9.5% B. vietnamiensis 3 1% B.cocovenenans 3 1% * Isolations on SP-G semi-selective medium (Tsushima et al., 1986).
26 Table 2.2.3. Results of BiologTM identification tests and seedling and panicle inoculation tests to determine pathogenicity of bacterial isolates from bacterial panicle blight infected rice collected in Louisiana. Bacterial species Pathogenicity Tests* Isolate number according to BiologTM seedling panicle 99gr-8-a B. glumae ++ +++ 99gr-2-a B. glumae +++ +++ 98gr-5 B. multivorans +++ +++ 99gr-3-b B. glumae ++ +++ 99gr-4-a B. glumae ++ +++ 6-1 P. resinovorans NP NP 98gr-1 B. gladioli NP NP 98gr-8-a P. tolaasii NP NP 99sh-2 B. multivorans + + 99gr-11 B. glumae +++ +++ 99gr-8-b No ID NP NP 98gr-12-a B. glumae ++ ++ 99gr-6-a B. glumae +++ ++ 99sh-6 B. glumae +++ +++ 80-1 B. glumae +++ +++ 98gr-13-b B. gladioli +++ + 99sh-9 B. glumae + ++ 32-5 P. putida NP +/NP** 99sh-18 B. glumae ++ +++ 101gr-2 B. glumae + ++ 99sh-3 B. glumae +++ +++ 99gr-2-b B. glumae +++ +++ 99gr-4-b B. glumae +++ +++ 99gr-7-a B. plantarii +++ +++ 99sh-15-a B. multivorans +++ +++ 99sh-12 B. multivorans ++ ++ 101gr-1 B. glumae +++ ++ 98gr-6-a B. glumae ++ ++ P. putida biotype 15R-1 NP A NP 99sh-16 B. plantarii +++ +++ 99gr-5-b B. multivorans NP ++ *NP = not pathogenic, ratings represent degree of virulence when strain was pathogenic with + = weakly virulent, ++ = moderately virulent, and +++ = highly virulent. ** NP/+ = NP or + on different inoculated plants.
27 Table 2.2.3. (continued) Bacterial species according Pathogenicity Tests* Isolate number TM to Biolog seedlings panicles 98gr-6-b B. glumae +++ ++ 99sh-10 B. gladioli +++ +++ 99sh-5 B. glumae ++ +++ 34-1 P. putid biotype A NP NP 98gr-2 P. putida NP NP 98gr-3 B. multivorans NP ++ 99sh-11 B. glumae +++ +++ 99sh-17 B. multivorans ++ +++ 98gr-13-a B. gladioli NP + 99gr-1-b B. multivorans ++ ++ 99gr-6-b B. glumae ++ +++ 50-9 P. aurantiaca NP NP 99gr-1-a B. glumae +++ +++ 99sh-7 B. gladioli NP NP 99sh-15-b B. multivorans +++ +++ 99gr-9 B. gladioli ++ +++ 99gr-4-b B. gladioli +++ +++ 99gr-3-a A. dispar NP NP 99gr-7-b B. glumae ++ +++ 11sh-5 B. multivorans NP NP 35-2 P. putida NP NP 25-2 P. aeruginosa NP NP 99gr-5-a B. glumae ++ + 10-4 S. maltophilia NP NP 99sh-14 B. glumae +++ +++ 98gr-12-b B. glumae ++ ++ 99sh-4-a B. vietnamiensis ++ +++ 99gr-4-b P. tolaasii +++ +++ 170gr-4 A.Tumefaciens/Radiobacter NP NP 191gr-6 B. plantarii ++ ++ 146sh-3 P. boreopolis NP NP *NP = not pathogenic, ratings represent degree of virulence when strain was pathogenic with + = weakly virulent, ++ = moderately virulent, and +++ = highly virulent. ** NP/+ = NP or + on different inoculated plants.
28 Table 2.2.3. (continued) Bacterial species Pathogenicity Tests* according to Isolate number BiologTM seedlings panicles 167gr-1 P. spinosa + ++ 189sh-7 B. multivorans ++ ++ 192gr-2 B. gladioli NP NP 191gr-1 V. fluvialis NP NP 189sh-1 A. a. ss avenae NP NP 190gr-4 P. aeruginosa NP NP 133sh-3 P. resinovorans NP NP 191sh-10 B. glumae NP +++ 127sh-7 P. spinosa NP NP 189gr-8 B. glumae NP NP 171gr-2 B. glumae + ++ 190gr-1 B. glumae + +++ 191sh-1 B. multivorans NP NP 156sh-1 P. resinovorans NP NP 157sh-1 B. multivorans + + 158sh-2 P. resinovorans NP NP 154sh-1-a B. plantarii ++ +++ 127sh-6 P. spinosa NP NP 170sh-1 B. gladioli + +/NP* 190gr-2 B. gladioli ++ ++ 156sh-1-a P. resinovorans NP NP 171gr-1-a B. gladioli ++ ++ 191gr-4 B. gladioli + + 170gr-3-a B. gladioli ++ ++ 190gr-3 B. gladioli ++ ++ 166gr-6 P. aurantiaca NP NP 171gr-3 B. gladioli ++ +++ 189gr-9 B. gladioli ++ ++ 191sh-2 P. corrugata NP NP 191sh-12 B. glumae ++ +++ 166gr-1 B. gladioli NP +++ 189gr-4 B. glumae +++ +++ 189gr-2 B. gladioli ++ ++ *NP = not pathogenic, ratings represent degree of virulence when strain was pathogenic with + = weakly virulent, ++ = moderately virulent, and +++ = highly virulent. ** NP/+ = NP or + on different inoculated plants.
29 Table 2.2.3. (continued) Bacterial species Isolate number according to Pathogenicity Tests* BiologTM seedlings panicles 189sh-6 A. a. ss avenae NP NP 147sh-2 A. a. ss cattleyae NP NP/+** 190gr-1 B. glumae + +++ 167sh-3 H. seropedicae NP NP 188gr-1 B. plantarii NP + 188gr-2 B. glumae ++ +++ 192gr-1 B. gladioli + +++ 166gr-2 B. gladioli ++ ++ 184sh+gr-2 S. maltophilia NP NP 191gr-2 B. plantarii ++ +++ 180gr-2 F. oryzihabitans -*** NP 154sh-1-b S. macrogoltabidus NP NP 171gr-1-b B. gladioli ++ ++ 156sh-1-b B. glumae + + 170gr-3-b B. gladioli +++ ++ #5: 193gr-1 B. multivorans + +++ 213sh-2 P. putida biotype B NP NP 198gr-2 P. tolaasii + +++ 201gr-1 B. multivorans NP +++ 192gr-7 B. plantarii ++ ++ 206gr-3 B. plantarii + ++ 207gr-2 B. multivorans + ++ 217gr-1 B. multivorans ++ +++ 210gr-1 P. agarici NP NP 202gr-2 B. plantarii + ++ 203gr-2 B, multivorans NP +++ 209gr-2 B. gladioli + +++ 201sh-1 B. multivorans NP NP 195gr-7 B. plantarii + +++ 196gr-4 B. multivorans + ++ 216gr-3-a B. multivorans + +++ 197gr-1-a B. multivorans ++ ++ 216gr-8 B. multivorans ++ +++ 200gr-4 P. tolaasii +++ +++ P.fluorescens 200gr-5 biotype G NP +++ *NP = not pathogenic, ratings represent degree of virulence when strain was pathogenic with + = weakly virulent, ++ = moderately virulent, and +++ = highly virulent. ** NP/+ = NP or + on different inoculated plants. *** - = no data available
30 Table 2.2.3. (continued) Bacterial species Pathogenicity Tests* Isolate number according to BiologTM seedlings panicles 208gr-3 B. multivorans ++ +++ 196gr-5 B. multivorans ++ +++ 210gr-3 B. gladioli +++ +++ 198gr-3 B. multivorans + +++ 216gr-3-b B. multivorans +++ ++ 209gr-1 B. multivorans +++ +++ 201gr-2 B. gladioli + +++ 216sh-1 B. gladioli + +++ 217gr-2 B. multivorans ++ +++ 207gr-4 B. gladioli ++ NP 206sh-1 P. fluorescens +++ ++ 192gr-5 B. plantarii + +++ 197gr-2 B. multivorans ++ +++ 195gr-11 B. glumae ++ ++ 197gr-1-b B. multivorans + +++ 213gr-1 P. fluorescens + +++ 195gr-5 B. multivorans ++ +++ 216gr-1 B. glumae ++ ++ 199gr-2 B. multivorans NP ++ 193gr-5 B. multivorans ++ +++ 196gr-1 B. multivorans NP +++ 200gr-3 B. multivorans NP +++ 216gr-6 B. multivorans + +++ 217gr-3 B. plantarii ++ ++ 203gr-1 B. multivorans NP ++ 195gr-6 B. plantarii NP +++ 201gr-2 B. multivorans + +++ P. fluorescens 210gr-4 + biotype G +++ 200sh-1 B. multivorans ++ +++ #6: 221gr-3 +++ +++ 237gr-3 B. gladioli +++ +++ 217sh-1 B. gladioli + ++ 252gr-5-b B. gladioli +++ +++ 250sh-1 B. gladioli +++ +++ 221sh-1 B. multivorans NP ++ 219sh-4 B. gladioli +++ +++ *NP = not pathogenic, ratings represent degree of virulence when strain was pathogenic with + = weakly virulent, ++ = moderately virulent, and +++ = highly virulent. ** NP/+ = NP or + on different inoculated plants.
31 Table 2.2.3. (continued) Bacterial species according Pathogenicity Tests* Isolate number TM to Biolog on seedlings on panicles 241gr-1 B. gladioli +++ +++ 220gr-1-b B. glumae ++ +++ 250gr-1 B. gladioli NP ++ 249sh-1 B. gladioli + + 218sh-2 B. glumae ++ ++ 222gr-1-a B. gladioli ++ +++ 227gr-2-a B. gladioli +++ +++ 220gr-2-b B. gladioli ++ ++ 250gr-2 B. plantarii ++ ++ 237gr-1 B. gladioli +++ +++ 227gr-1 B. gladioli +++ +++ 228gr-1 B. gladioli +++ +++ 221gr-1 B. multivorans +++ +++ 252gr-5-a B. multivorans +++ +++ 222gr-2 P. syringae pv zizanize ++ +++ 230sh-1 P. pyrrocinia ++ ++ 252gr-2 B. gladioli + ++ 237gr-5 B. gadioli NP NP 235gr-2 B. gladioli ++ ++ 218gr-1 B. glumae +++ ++ 252gr-1 B. gladioli +++ +++ 227gr-2-b B. glumae +++ +++ 252gr-7 B. multivorans ++ + 222gr-1-b B. glumae + + 249gr-2 B. gladioli +++ ++ 230gr-1 B. gladioli +++ ++ 241gr-3 B. gladioli +++ +++ 218sh-1 B. gladioli ++ +++ 236gr-1 B. gladioli ++ ++ 243gr-1 B. gladioli ++ ++ 249gr-3 P. tolaasii ++ +++ 223gr-1 B. gladioli ++ ++ 219sh-2 P. syringae pv tagetis NP ++ 238gr-3 B. gladioli +++ ++ 236gr-3 B. gladioli +++ ++ *NP = not pathogenic, ratings represent degree of virulence when strain was pathogenic with + = weakly virulent, ++ = moderately virulent, and +++ = highly virulent. ** NP/+ = NP or + on different inoculated plants.
32 Table 2.2.3. (continued) Bacterial species Pathogenicity Tests* Isolate number TM according to Biolog seedlings panicles 235gr-1 B. gladioli ++ + +++ 252gr-4 B. plantarii ++ +++ 252gr-2 B. gladioli +++ +++ 219sh-1 B. gladioli ++ +++ 230sh-2 B. gladioli +++ +++ 235sh-1 B. multivorans +++ +++ 220gr-1-a P. pyrrocinia +++ +++ 223gr-2 B. gladioli ++ +++ 220gr-2-a B. gladioli +++ +++ 218gr-3 B. plantarii + ++ 243gr-3 B. gladioli +++ +++ 226gr-1 B. glumae +++ +++ 237gr-5 B. gladioli NP NP #7: 258gr-5 B. multivorans +++ ++ 261gr-9 B. multivorans NP +/NP 253gr-2 A. facilis NP NP 261gr-1 B. multivorans +++ +++ 258gr-8 B. glumae +++ ++ 257gr-2 B. gladioli ++ +++ 257sh-1 B. gladioli NP NP 270sh-4 B. plantarii +++ +++ 273sh-1 B. multivorans +++ +++ 259gr-1 B. gladioli ++ ++ 253sh-1 B. multivorans +++ +++ 266gr-1 S. macrogoltabidus NP ++
256sh-2 R. radiobacter NP NP 256gr-2 B. multivorans NP + 261gr-2 B. multivorans ++ +++ 266gr-5 B. multivorans +++ +++ 259gr-4 B. gladioli NP ++ 261gr-5 V. tubiashii ++ ++ 273sh-2 B. multivorans + +++ 258gr-4 B. gladioli + ++ 254sh-4 A. avenae. ss cattleyae NP NP 266sh-2 P. tolaasii +++ +++ *NP = not pathogenic, ratings represent degree of virulence when strain was pathogenic with + = weakly virulent, ++ = moderately virulent, and +++ = highly virulent. ** NP/+ = NP or + on different inoculated plants.
33 Table 2.2.3. (continued) Bacterial species Pathogenicity Tests* Isolate number TM according to Biolog seedlings panicles 259gr-5 B. gladioli ++ ++ 261gr-4 B. multivorans +++ +++ 272gr-4 B. multivorans NP + 254sh-1 P. tolaasii +++ +++ 260gr-1 B. glumae + +++ 258gr-1 B. multivorans NP ++ 258gr-5 B. multivorans +++ ++ 256sh-3 P. tolaasii NP +++ 256gr-1 B. phenazinium + ++ 253sh-2 A. a ss cattleyae NP NP 255sh-1 P. tolaasii + ++ 269gr-1-a B. gladioli +++ ++ 257sh-2 B. glumae ++ +++ 257gr-1 C. meningosepticum NP ++ 254gr-1 B. gladioli + +++ 267gr-1-b S. sanguinis NP NP/+ 273gr-1 B. glumae + +++ 260gr-3 B. plantarii NP + 257gr-2 B. gladioli NP +++ 254gr-5 B. plantarii ++ +++ 254gr-3 F. ferrugineum NP NP/+ 259gr-3 B. plantarii + +++ 265gr-1 B. glumae + ++ 270sh-2 B. plantarii NP +++ 255gr-2 B. multivorans ++ +++ 273gr-4 B. glumae ++ + 267gr-2 B. plantarii ++ + 270gr-1 P. tolaasii ++ +++ 254gr-2 B. gladioli +++ +++ 269gr-1-b B. glumae ++ +++ 272gr-2 B. multivorans +++ +++ 106sh-11 B. glumae +++ +++ 396gr-2 B. gladioli NP NP 363gr-1 B. gladioli NP + 407gr-6 B. gladioli +++ +++ *NP = not pathogenic, ratings represent degree of virulence when strain was pathogenic with + = weakly virulent, ++ = moderately virulent, and +++ = highly virulent. ** NP/+ = NP or + on different inoculated plants.
34 Table 2.2.3. (continued) Bacterial species Pathogenicity Tests* according to Bacterial isolates BiologTM seedlings panicles 387gr-2-b B. gladioli +++ +++ 406gr-3 B. gladioli +++ +++ 408gr-4 B. cocovenenans +++ +++ 357-8 B. gladioli ++ ++ 366gr-1 B. multivorans +++ +++ 379gr-1-a-(1) B. glumae +++ +++ 106sh-7 B. vietnamiensis + ++ 378g-3 B. gladioli ++ + 106sh-9 B. plantarii NP NP 357gr-1 B. gladioli NP NP 106sh-5 B. glumae NP NP 384gr-1 P. viridilivida NP NP 395-2 B. gladioli NP NP 408gr-7 B. gladioli + ++ 403gr-2 B. glumae ++ +++ 980007-1(5) B. glumae +++ +++ 405gr-4 B. cocovenenans +++ +++ 961149-4(4) B. gladioli NP NP 407gr-3 B. plantarii +++ +++ 357-3 B. gladioli ++ + 106gr-3 B. glumae ++ + 106sh-4-b B. glumae +++ +++ 387gr-2-a B. glumae ++ +++ 367gr-1 B. glumae ++ +++ 395gr-2 B. gladioli +++ +++ 367gr-3 B. glumae ++ +++ 254gr-2 B. multivorans +++ +++ 255gr-2 B. glumae ++ ++ 373gr-1 B. gladioli +++ +++ 318gr-4 B. glumae ++ +++ 366gr-2 B. gladioli NP NP 318gr-1 B. multivorans +++ +++ 379gr-1-b B. gladioli NP NP 980007-1(6) B. gladioli +++ +++ *NP = not pathogenic, ratings represent degree of virulence when strain was pathogenic with + = weakly virulent, ++ = moderately virulent, and +++ = highly virulent. ** NP/+ = NP or + on different inoculated plants.
35 Table 2.2.3. (continued) Bacterial species Pathogenicity Tests* Isolate number TM according to Biolog seedlings panicles 382gr-1 B. gladioli +++ ++ 385gr-1 B. gladioli ++ + 106sh-4-a B. glumae NP NP 366gr-6 B. vietnamiensis ++ ++ 398gr-3 B. glumae +++ +++ 373gr-2 P. viridilivida NP NP 398gr-1 B. glumae +++ +++ 407gr-8 B. gladioli + ++ 407gr-1 B. gladioli +++ +++ 379gr--1-a-(2) B. gladioli ++ ++ 951886-4(3) B. glumae ++ +++ 372gr-1 B. gladioli ++ +++ 379gr-1-a-(1) B. multivorans ++ ++ 393gr-7 P. tolaasii - NP/+** 321gr-9 B. gladioli + + 321gr-2-a B. gladioli +++ + 321gr-3 B. gladioli ++ ++ 951886-4(3) B. gladioli +++ ++ 321gr-6 B. gladioli ++ + 325gr-1 B. gladioli + + 321gr-8-a B. gladioli ++ + 324gr-2 B. gladioli ++ +++ 324gr-3 B. gladioli ++ +++ 319gr-4 P. syringae pv zizanize ++ + 321gr-2-b B. gladioli ++ ++ 321gr-4 B. plantarii ++ ++ 325gr-2 B. gladioli ++ ++ 321gr-8-b B. gladioli ++ ++ 325gr-4 B. gladioli ++ +++ 321gr-7 B. gladioli ++ ++ 319gr-1 B. gladioli +++ +++ 319gr-6 - *** + + 154sh-1-a B. gladioli + ++ *NP = not pathogenic, ratings represent degree of virulence when strain was pathogenic with + = weakly virulent, ++ = moderately virulent, and +++ = highly virulent. ** NP/+ = NP or + on different inoculated plants. *** - = no data available
36 Table 2.2.3. (continued) Bacterial species Pathogenicity Test* Isolate number TM according to Biolog seedling panicle 113sh2-7 B. glumae P +++ 113sh2-4 B. plantarii P ++ 113sh2-5 B. glumae P + 113sh2-2-a B. glumae P + 113sh2-8 B. glumae P +++ 117sh1-3 B. glumae P +++ 117sh1-8 B. glumae P +++ 117sh2-3 B. glumae P +++ 117g1-7-a B. glumae NP NP 116g1-1 B. glumae P +++ 117g1-8 B. glumae P +++ 117g1-9 B. glumae P +++ 117sh1-1 S. maltophilia NP NP 120g1-1 B. glumae P ++ 119g1-5 B. glumae P +++ 123sh-3 - - - 118sh-6 A. avenae ss cattleyae NP NP 119g1-4 B. glumae NP ++ 119g1-3 P. pyrrocinia P +++ 120g1-2 B. glumae P +++ 122sh-1 - NP NP 117g1-11 B. glumae P +++ 118sh-5 P. group2 (B.-like) NP NP 114sh1-4 B. cepacia P NP 122sh-5 A. jandaei DNA group 9 NP NP 113sh2-9 B. glumae P +++ 113sh2-3 B. plantarii P NP 113sh1-10 C. acidovorans NP NP 124sh-1 - - - 124sh-4 - - - 113sh2-2-b - P + 117g1-7-b - P ++ *NP = not pathogenic, ratings represent degree of virulence when strain was pathogenic with + = weakly virulent, ++ = moderately virulent, and +++ = highly virulent. ** NP/+ = NP or + on different inoculated plants.
37 Table 2.2.4. The frequency of pathogenic bacterial strains of each species isolated from rice based on inoculation of Cypress rice plants. Number of Pathogenic Total number of Relative pathogenic bacterial isolates of each frequency isolates of each species species (%) identified species Burkholderia spp. B. glumae 62 68 91 B. gladioli 85 103 83 B. multivorans 44 60 73 B. plantarii 20 25 80 B. cocovenenans 3 3 100 B. vietnamiensis 3 3 100 Pseudomonas spp. P. syringae pv zizanize 2 2 100 * P. fluorescens (biotype G) 3 4 75 P. tolaasii 7 12 58 P. pyrrocinia 3 3 100 P. spinosa 1 3 33 Vibrio spp. V. tubiashii 1 1 100 * P. fluorescens or P. fluorescens biotype G
2.2.2 Pathogenicity Tests and Symptoms Produced Pathogenicity tests were applied using the method of injection of bacterial suspensions into the stems of 4-leaf-stage rice seedlings 1 – 2 days before tillering or into boots just before panicles emerged using the variety Cypress. Continuous observations and records for the progressive symptoms were made with a digital camera and notes were taken for 20 – 25 days after inoculation. A range of leaf and leaf sheath symptoms was observed on seedlings (Figures 2.2.6, 2.2.7, 2.2.8, and 2.2.9) and the boot leaf sheath ( Figure 2.2.11, 2.2.18, and 2.2.20), emerging panicles including poor panicle emergence ( Figure 2.2.10) grain discoloration, sterile spikelets, and complete blacking of hulls (Figure 2.2.19). Panicle damage and grain discoloration existed in various degrees of severity. In the severest case, a poor or total lack of panicle emergence occurred. The lesions on sheaths developed longitudinally into with brown/black necrosis on sheaths, with grayish-white centers and dark brown, distinct margins. Symptoms produced by identified B. glumae cultures were similar among all pathogenic Burkholderia species. In infections with intermediate virulence, a restricted foci around the injection point was
38 formed on the sheath (Figure 2.2.8). For inoculated rice seedlings, a widespread symptom was yellow or light-brown rot of the leaf sheath. The development of the disease depended strongly on weather conditions. Cooler temperatures of 23oC – 25oC significantly reduced lesion development on affected leaf sheaths. No infection was expressed as a yellow or small light brown area around the injection site (Figure 2.1.3). An experiment where seedlings and emerging panicles were simultaneously inoculated with the same bacterial strains under, under cool temperature conditions, confirmed that unlike rice seedlings, emerged panicles were still severely damaged. In a few cases, B. plantarii and B. cocovenenans expressed distinct symptoms. A brown leaf stripe along mid-veins was produced by B. plantarii (Figure 2.2.7) and with B. cocovenenans leaf discoloration spread along the leaf blade edge to the tip of the leaf (Figure 2.2.9). However, whether symptoms can be used to distinguish pathogenic strains needs further study. Typical infection by virulent or moderately virulent strains of several species of Burkholderia are shown in Figures 2.2.12 to 2.2.17.
Figure 2.2.6. Leaf sheath rot on Cypress seedlings caused by B. glumae isolate106sh-4-b on the left and B. gladioli isolate 980007-1(6) on the right. Please note that the whole inoculated leaf sheath rots when the bacterial isolate is highly virulent.
39
Figure 2.2.7. Brown and tan leaf stripes or lesions on seedling leaf blades infected by B. glumae isolate 106sh-11on the left and B. plantarii isolate 407gr-3 on the right.
Figure 2.2.8. Low virulence reaction produced by B. gladioli (isolate 357gr-1 on the left) and B. gladioli (isolate 406gr-3 on the right). Symptoms were expressed as a brown lesion of less than 2cm length around the inoculation site with a distinct darker brown margin.
40
Figure 2.2.9. Leaf discoloration extending along the edge of the leaf blade down to the leaf tip caused by B. cocovenenans (isolate 405gr-4 on the left) and the uninoculated Cypress control on the right.
Figure 2.2.10. Poor emergence of panicle with spilelet sterility by caused by B. multivorans isolate 366gr-1on the left and a dark brown sheath and grain Rot caused by B. glumae isolate 367gr-3 on the right.
41
Figure 2.2.11. Extended brown necrotic lesion caused by a highly virulent strain of B. glumae (367gr-3 on the left) and a dark brown lesion surrounding the inoculation site on the sheath caused by the moderately virulent B. glumae strain (398gr-3 on the right).
Figure 2.2.12. Strain 98gr-5 of Burkholderia multivorans showing a virulent (+++) reaction on Cypress rice after seedling inoculation.
42
Figure 2.2.13. Strain 98gr-5 of Burkholderia tolaasii, on Cypress rice showing a highly virulent (+++) reaction after seedling inoculation.
Figure 2.2.14. Seedling disease reaction (+++) caused by bacterial strain 99gr-2-b of Burkholderia glumae on Cypress rice.
43
Figure 2.2.15. Highly virulent seedling disease reaction (+++) of bacterial strain 99sh-10, Burkholderia gladioli on Cypress rice.
Figure 2.2.16. Highly virulent seedling disease reaction (+++) of bacterial Strain 99sh-16, Burkholderia plantarii on Cypress rice.
44
Figure 2.2.17. Seedling disease ++ reaction of bacterial strain 99sh-4-a, Burkholderia vietnamiensis on Cypress rice.
Figure 2.2.18. Highly virulent panicle and flag leaf sheath reaction (+++) on isolate 210gr-3 of Burkholderia gladioli on Cypress rice after boot inoculation (overview).
45
Figure 2.2.19. Close-up view of severe panicle blighting symptoms on Cypress rice resulting from inoculation with isolate 210gr-3 of Burkholderia gladioli.
46
Figure 2.2.20. Close-up view of flag leaf sheath lesions produced when Cypress rice boots were inoculated with isolate 210gr-3 of Burkholderia gladioli.
2.2.3 Colony and Culture Characteristics of the Bacterial Pathogens Examinations of colony morphology showed that the Burkholderia species could not be distinguished simply by colony or culture characters. All of the species formed convex colonies with smooth margins or flat colonies with pleated margins on KBA medium, releasing yellow pigment or no pigment into the medium. Most cultures had gray-white or cream-white colonies. In a very few cultures there was a reddish tint to the colonies. Yellow or gray-white colonies were the most common. B. gladioli colonies were yellow, gray-white, or yellow-green in color, but all of these colony-types were pathogenic to rice plants, suggesting that there may be toxins other than the phytotoxin associated with the yellow pigment. All avirulent colonies of B. glumae, B. gladioli, B. multivorans and B. plantarii were gray-white or cream white colonies that did not release
47 pigments into the medium (Figures 2.2.21, 2.2.22, and 2.2.23). When selected cultures of B. glumae and B. gladioli were grown on King’s B medium and observed for production of pigment (toxin) in the medium, it was clear that cultures that produced the yellow or green pigments were pathogenic and virulent (Table 2.2.5). Cultures that did not produce pigment were avirulent. Several non-pathogenic and non-pigment producing cultures of both B. glumae and B. gladioli were identified in this experiment. These cultures will be useful for future experiments to determine what controls toxin production and if this character can be transferred from virulent cultures to avirulent cultures. It’s possible that the gene(s) for toxin production are carried by a plasmid that can be transferred among Burkholderia spp.
Figure 2.2.21. Cultures of B. plantarii, virulent strain (left) and avirulent strain (right). Note the yellow toxin in the virulent culture and the lack of yellow toxin in the medium of the avirulent strain.
48 Table 2.2.5. Production of yellow or green pigment (toxin) in King’s B medium by pathogenic and nonpathogenic Burkholderia glumae and Burkholderia gladioli isolates. Seedling Panicle Pigment Isolate Number Bacterial species pathogenicity pathogenicity (toxin)* rating rating 357gr-1 B. gladioli NP NP N 106sh-5 B. glumae NP NP N 395-2 B. glumae NP NP N 366gr-2 B. gladioli NP NP N 237gr-5 B. gladioli NP NP N 99sh-7 B. gladioli NP NP N 257sh-1 B. gladioli NP NP N 321gr-9 B. gladioli + + W 379gr-1-b B. gladioli NP NP N 396gr-2 B. gladioli NP NP N 98gr-13-a B. gladioli NP + N 259gr-4 B. gladioli NP ++ W 363gr-1 B. gladioli NP + N 189gr-8 B. glumae NP NP W 191sh-10 B. glumae NP +++ W 250gr-1 B. gladioli NP ++ W 321gr-9 B. gladioli + + W 192gr-2 B. gladioli NP NP W 166gr-1 B. gladioli NP +++ IM 98gr-1 B. gladioli NP NP N 106sh-4-a B. glumae NP NP N 319gr-1 B. gladioli +++ +++ G 398gr-1 B. glumae +++ +++ I 106sh-11 B. glumae +++ +++ I 980007-1(5) B. glumae +++ +++ I 321gr-2-a B. gladioli +++ + IM 406gr-3 B. gladioli +++ +++ I 382gr-1 B. gladioli +++ ++ IM 407gr-1 B. gladioli +++ +++ I 99gr-6-a B. glumae +++ ++ I 99gr-2-a B. glumae +++ +++ I * Production of pigment (toxin) is indicated as “I” = intense, “IM” = intermediate, “W” = weak (very faint color produced), and “N” = none. “G” = no yellow pigment produced, but green pigment was produced.
49
Figure 2.2.22. Cultures of Burkholderia glumae with a virulent strain on the left and an avirulent strain on the right. Note the lack of toxin in the medium on the right.
Figure 2.2.23. Cultures of Burkholderia gladioli, virulent strain (left) and avirulent strain (right). Note the lack of yellow pigment (toxin) in the medium on the right and the presence of yellow pigment on the left.
2.2.4. Clustering of Bacterial Strains Isolated from Rice Showing Panicle Blight into Functional Groups In order to compare the similarity of strains of the different species to representative strains stored in the BiologTM GN database, bacterial isolates were clustered into functional groups based upon data from BiologTM identification tests and pathogenicity tests. Each isolate was therefore assigned two characteristic parameters: a virulence parameter and a typicality or similarity parameter. Evaluation using the
50 virulence system produced four groups: non-virulent strains, low virulence strains, middle-virulent strains, and high virulence strains. Evaluation using the similarity system formed two groups: low similarity strains and high similarity strains (Table 2.2.6). The threshold value of high / low similarity of B. plantarii was assigned at 0.6. Considering the larger number of strains in B. glumae, B. multivorans and B. gladioli, a weight was added to the threshold values; shifting the values of B. multivorans (Table 2.2.7) and B. glumae to 0.65 and B. gladioli strains were shifted to 0.7. The results indicated that B. glumae (Table 2.2.9) and B. gladioli (Table 2.2.8) possessed more virulent strains and higher similarity indexes as strain populations than that of B. plantarii, suggesting these two species may be the main pathogens in this study. A higher similarity index implied higher detection accuracy and high similarity to reference strains. However, low similarity might not mean low detection accuracy, but imply that they were different types, considering that bacterial metabolic types are variable (Jones, 1993). B. multivorans expressed an intermediate pattern of population distribution between B. plantarii, B. gladioli, and B. glumae according to the above evaluation system, suggesting that they took second place in the importance of pathogens (Table 2.2.7).
Table 2.2.6. Clustering of Burkholderia plantarii into functional and typical strains. High Virulence* Low Virulence Non-Virulent Mid Virulence High Low High Low High Low High Low similarity** similarity similarity similarity similarity similarity similarity similarity
407gr-3 195gr-7 206gr-3 260gr-3 195gr-6 217gr-3 192gr-7 270sh-4 154sh-1- 192gr-5 202gr-2 188gr-1 270sh-2 252gr-4 a 99sh-16 218gr-3 321gr-4 217gr-3 99gr-7-a 259gr-3 191gr-2 254gr-5 267gr-2 191gr-6 250gr-2 * The three-scale system of disease severity was determined by pathogenicity tests. ** A threshold value of similarity to typical strains was assigned with a similarity index of 0.6 in this table. The similarity was considered as high similarity if the index was more than 0.6; otherwise as low similarity.
51
Table 2.2.7. Clustering of Burkholderia multivorans into functional and typical strains.* High Virulence Low Virulence Non-Virulent Mid Virulence High Low High Low High Low High Low similarity similarity similarity similarity similarity similarity similarity similarity 98gr-5 366gr-1 198gr-3 99sh-2 258gr-1 11sh-5 252gr-7 99gr-1-b 261gr-4 318gr-1 201gr-2 273sh-2 261g-9 99gr-5-b 216gr-8 99sh-17 99sh-15- 197gr-1- 197gr-1- 261gr-1 157sh-1 221sh-1 272gr-4 99sh-12 a b a 216gr-3- 273sh-1 266gr-5 196gr-4 201sh-1 256gr-2 196gr-5 261gr-2 a 252gr-5- 253sh-1 193gr-1 203gr-1 191sh-1 208gr-3 255gr-2 a 216gr-3- 272gr-2 207gr-2 196gr-1 201gr-1 379gr-1 200sh-1 b 209gr-1 235sh-1 216gr-6 200gr-3 203gr-2 a(1) 217gr-1 254gr-2 199gr-2 217gr-2 258gr-5 197gr-2 221gr-1 195gr-5 193gr-5 * A threshold value of similarity to typical strains was assigned with a similarity index of 0.65 in this table
52 Table 2.2.8. Clustering of Burkholderia gladioli into functional and typical strains.*
High Virulence Low Virulence Non-Virulence Mid Virulence
High Low High Low High Low High Low similarity similarity similarity similarity similarity similarity similarity similarity
321gr-2- 379gr-1- 325gr-1 408gr-7 321gr-9 321gr-6 99gr-9 a b 99sh-10 220gr-2- 319gr-1 191gr-4 192gr-1 166gr-1 99sh-7 325gr-4 b 170gr-3- 98gr-13- 170sh-1 209gr-2 237gr-5 98gr-1 321gr-3 235gr-1 b b 254gr-2 98gr-13- 243gr-3 217sh-1 216sh-1 357gr-1 325gr-2 243gr-1 a 241gr-3 241gr-1 249sh-1 363gr-1 257sh-1 324gr-2 219sh-1 249gr-2 219sh-4 258gr-4 396gr-2 257gr-2 324gr-3 166gr-2 252gr-2 321gr-8- 237gr-3 254gr-1 366gr-2 192gr-2 207gr-4 a 252gr-5- 321gr-8- 170gr-3- 237gr-1 407gr-8 250gr-1 b b a 227gr-2- 236gr-3 154sh-1- 321gr-2- 259gr-4 235gr-2 a a b 252gr-1 228gr-1 321gr-7 99gr-4-a 210gr-3 171gr-1- 230sh-2 b 250sh-1 227gr-1 171gr-3 220gr-2- 230gr-1 189gr-2 a
269gr-1- 190gr-2 a 238gr-3 190gr-3 382gr-1 189gr-9 980007- 171gr-1-
1(6) a 407gr-6 223gr-1 406gr-3 223gr-2 222gr-1- 407gr-1 a 395gr-2 387gr-2- 236gr-1
b 218sh-1 259gr-5 259gr-1 372gr-1 378gr-3 385gr-1 379gr-1-
a(2) 357-8 357-3 *A threshold value of similarity to typical strains was assigned with a similarity index of 0.70 in this table.
53 Table 2.2.9. Clustering of Burkholderia glumae into functional and typical strains.* High Virulence Low Virulence Non-Virulent Mid Virulence
High Low High Low High Low High Low similarity similarity similarity similarity similarity similarity similarity similarity 252gr-5- 156sh-1- 189gr-4 171gr-2 189gr-8 106sh-5 191gr-2 a b 222gr-1- 216gr-1 218gr-1 258gr-8 265gr-1 191sh-10 188gr-2 b 195gr-11 106sh-4- 189sh-7 226gr-1 99sh-6 260gr-1 99sh-9 218sh-2 a 273gr-4 227gr-2- 220gr-1- 99gr-11 273gr-1 101gr-2 395-2 b b 98gr-12- 80-1 98gr-6-b 190gr-1 257sh-2 b 106sh-4- 269gr-1- 101gr-1 99gr-7-b b b 99gr-6-a 398gr-3 99gr-8-a 99sh-18 99gr-2-a 99gr-3-b 99gr-5-a 99gr-4-b 99sh-14 99sh-5 367gr-1 951886 99gr-2-b 98gr-6-a -4(3) 99sh-3 99gr-6-b 98gr-12- 99sh-11 a 99gr-1-a 318gr-4 99sh-15- 367gr-3 b 398gr-1 403gr-2 980007- 255gr-2 1(5) 387gr-2- 106sh-11 a 387gr-2- 106gr-3 b * A threshold value of similarity to typical strains was assigned with a similarity index of 0.65 in this table.
Based on the percentage of isolates of each Burkholderia species among the bacterial isolates from rice showing BPB symptoms, B. gladioli, B. glumae, and B. multivorans appear to be the most important species causing BPB in Louisiana rice (Table 2.2.24).
54 120 The Number of Isolates 100 80 Relative Frequency (%) 60 40 20 0
e i ii a iol s r s s d ta si m a ran n n lu gl la e nan . ivo mi e . g B lt . p a n B B n ve t o mu e c B. vi o . .c B B
Figure 2.2.24. The Frequency of distribution of Burkholderia species isolated from rice plants (Oryza sativa L. cv. Cypress) showing BPB symptoms.
18 16 ates
l 14 o
s 12 I f
o 10
er 8 b
m 6 u 4 e N
h 2 T 0 i i i i i im im im im im imi -s -s s -s -s s h h- g w- g g ow o Low i L Hi L Hi H + + + u+ u ru ir ru+ ir i iru+ iru i v -v v -v -v v - - h h w on ig w- on g Lo N N Hi H Lo
Figure 2.2.25. Functional classification of bacterial isolates from rice plants showing BPB symptoms and identified as B. glumae.
Based on the similarity/pathogenicity groupings, it looks like B. glumae (Figure 2.2.25) and B. gladioli (Table 2.2.26) may have races based on the distribution of pathogenicity and closeness of relatedness.
55 25
ates 20 l o s I
f 15 o er
b 10 m u
N 5 e h T 0 i i i i i i im im im m im im s -s s -si -s -s h- ig w igh- ig ow H o H Low H L + L + u + u u+ ru+ ir ru ir ir iru+ i v i -v v -v - -v -v h - n n h g w w o g Lo N No Hi Hi Lo
Figure 2.2.26. Functional classification of the bacterial isolates from rice plants showing BPB symptoms and identified as B. gladioli.
2.3 Summary and Conclusions Several genera and species of bacteria were isolated from Louisiana-grown rice showing symptoms of panicle blighting. Thirty nine bacterial species in 10 genera could grow on the semi-specific medium, SP-G, suggesting that this medium was not unique for selecting B. glumae and thus the species growing on the medium partly represented the general distribution of bacteria in diseased rice tissues. Among the Burkholderia isolates B. gladioli and B. glumae had the largest numbers of isolates highly virulent to Cypress rice. This suggested that they were the species in the Burkholderia complex most important in causing BPB when compared to B. multivorans, B. plantarii, and other isolated Burkholderia and pathogenic Pseudomonas species. The components of populations and geographic distributions of bacterial pathogens are determined by many factors, including weather conditions, cultivar and lines, root location, and soil types. The cultivar Cypress was previously reported to be susceptible to B. glumae (Shahjahan et al., 2000b). Whether the cultivar was equally susceptible to pathogenic Pseudomonas spp. needs further study. Soil type is an important factor affecting not only the components of populations of plant-associated bacteria, but also the variability of genetics in bacteria. It has been shown that the B. cepacia complex displays a wide variability in genetics. Dalmastri et al. (1999)studied the associations between the genetic diversity of maize root-associated B. cepacia
56 populations with soil type and the cultivars grown. They confirmed that soil type played a more important role in determining the genetic diversity of B. cepacia populations than did the cultivars grown. B. multivorans, B. gladioli, and B. glumae were found to be the most common isolates from panicle blighted rice plants in Louisiana in this study. The effect of soil types, cultivars, weather, and other elements affecting population dynamics of the complex needs further study. The fact that many non-pathogenic Psudomonas species were isolated from diseased tissue needs further exploration, including an evaluation of the roles of Pseudomonas species in the process of development of the syndrome and ecological relationships with the pathogenic bacteria. B. glumae, B. gladioli, B. multivorans, and B. plantarii strains, isolated from diseased rice in the southern United States, caused flag leaf sheath lesions, spikelet sterility, and poor emergence of panicles after inoculation. In most cases, they induced discoloration and rot of leaf sheaths on rice seedlings, consistent with the literature. Similar symptoms were reported for P. fuscovaginae causing brown-black necrosis on stems and poor emergence of panicles (Rott et al., 1989), suggesting that some functional characteristics, including pathogenicity, are similar to each other even though they belong to different genera and tend to have different geographic distributions. P. syringae pv. aptata was confirmed to attack developing florets of rice plants, producing similar early symptoms to B. glumae and P. fuscovaginae (Goto et al., 1987). Two pathovars of P. syringae were found in this investigation. P. syringae pv. zizanize was pathogenic and produced symptoms similar to those produced by Burkholderia spp. on both seedlings and panicles of rice. Variability of bacteria for pathogenicity is a universal phenomenon, with several possible causes including heterogeneity within strains, biochemical variability, variance of pathogens, and loss of disease agents during inoculation. This study showed that continued subculturing of isolates could cause loss of virulence of B. glumae. In this survey, there were 26 Burkholderia isolates expressing non-pathogenicity on seedlings, but pathogenicity on panicles inoculated at the late boot stage. The reverse only occurred once. It may be that weakly virulent strains can infect the more susceptible immature panicle tissues, but not the rapidly developing seedling tissues. As the BPB disease is
57 seedborne, avoiding contaminated seeds was essential for inoculation studies. No infection of non-inoculated rice at the seedling or panicle stages was observed in this study. Therefore, in this study the results of pathogenicity tests were mainly based on the experiments on seedlings in order to ensure the reliability of results. The BPB syndrome is poorly defined. The development of symptoms and severity of disease not only depends on virulence of the strain, but also on environmental factors, particularly weather conditions. Klement (1955) pointed out that P. fuscovaginae and P. syringae grow well in relative cool, wet weather. The frequency in Japan of the occurrence of the seedling rot disease caused by B. glumae, where the weather is cooler and drier than Louisiana, was due to the introduction of new planting methods where seedlings were grown in seedling boxes in warehouses maintained at high temperatures and humidity (Goto et al., 1987). Our research showed that a temperature range of 37oC - 41oC was a critical environmental factor affecting the occurrence and development of the disease in the greenhouse, not only for B. glumae, but also for B. gladioli, B. multivorans, and B. plantarii. Therefore, in inoculation tests the descriptions of typical symptoms and the determinations of disease severity only had significance when these environmental conditions were met. In this study, all of the identified isolates obtained an acceptable similarity index, over 0.5 as assigned by the BiologTM System, and the identities could therefore be reported according to “GN2 MicroPlateTM Instructions for Use” (Biolog Co., Hayward, CA ). The highest indexes were received by some B. gladioli strains, which reached up to 1.0, indicating a full match with a reference strain in the database library. The 4.20 version of BiologTM database used in this study has been greatly improved as more strains were added to the library, offering a powerful technique to support identification of bacteria. Some Pseudomonas species were identified to the pathovar level. However, the identification of bacteria is a long process and cross-referencing among two or more detection methods is desirable to ensure that an isolate can be identified accurately. Fatty acid analysis for the isolated reported on in this research is being made separately by our laboratory.
58 Furthermore, less than 0.5 similarity indexes may not necessarily mean low detection accuracy. It should be considered that some atypical strains have not been collected into the database. It should also be considered that natural variability among strains of the same bacterial species may give differences in metabolic types. There also may be loses of some biochemical capabilities during continuing culturing and storage of strains. An effective control measure for plant diseases is to eradicate sources of contamination with the pathogen. The BPB pathogens are seedborne in rice seeds harvested the previous year (Shahjahan et al., 1998b). B. glumae could not be detected from seeds kept outdoors for 5 months ((Tsusima et al., 1989), suggesting that low temperature treatment may kill the bacterium. Dry heat treatment using 65oC for 6 days or salt water selection could not ensure pathogen-free seeds in their studies. Monitoring the contamination of commercial seed-lots seems to be more practical than chemical control or using cold or heat treatments at this time based on current seed production, storage, and planting practices in Louisiana. A seed treatment pesticide to control BPB is not presently labeled for use in the United States. Two hundred and sixty two bacterial isolates from diseased rice tissues showing BPB symptoms were identified as Burkholderia species using the BiologTM GN2 test, comprising 75% of the identified bacteria. Six Burkholderia species were identified including B. glumae, B. gladioli, B. multivorans (B. cepacia genomovar II), B. plantarii, B. vietnamiensis (B. cepacia genomovar V) and B. cocovenenans. Of them, B. gladioli, B. multivorans, B. glumae, and B. plantarii comprised about 90 % of the Burkholderia isolates. Pathogenicity tests on seedlings and emerging panicles showed that six species were rice pathogens causing sheath rot or/and panicle blight. Eighty three percent of the B. gladioli, 91% of B. glumae, 73% of B. multivorans, and 80% of B. plantarii were pathogenic strains. It is therefore suggested that a complex of Burkholderia species caused the BPB syndrome recently found in Louisiana. These species caused seeding blight, seedling leaf sheath rot, and flag leaf sheath brown-black rot, sterile florets, grain discoloration, and/or poor emergence of panicles, similar to the symptoms reported in the literature. Symptom expression alone was not sufficient to distinguish Burkholderia pathogens to species level.
59 This appears to be the first report of B. multivorans as a pathogen on rice. Pathogenicity tests and data analysis showed that B. gladioli and B. glumae had more highly virulent strains than B. multivorans and B. plantarii, suggesting that they played the critical role in pathogenicity on rice plants. Morphology studies showed that convex colonies with smooth margins and yellow or gray-white in color on KB medium were common morphological characteristics among these species. Universally, avirulent strains were associated with gray-white colonies, supporting reports in the literature. Pseudomonas species were shown to be one of the causes of the syndrome. They included two P. syringae pv. zizanize, three P. fluorescens, three P. pyrrocinia, one P. spinosa and seven P. tolaasii strains, suggesting that Pseudomonas species could also be involved in the complex of bacterial species causing BPB on rice in Louisiana. The factors determining whether a given bacterium is the main casual agent may include weather conditions or cultivar and line susceptibility.
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Many papers were reviewed for this research that covered research that was indirectly related to the research area. As these papers were not cited, but contained information of use in future research with pathogenic bacteria on rice, they were listed in an Appendix as “BIBLIOGRAPHY OF RELATED LITERATURE”.
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96 VITA
Xianglong Yuan was born on Juan 12, 1962, in Shenyang, Liaoning, P. R., China, where he accomplished his elementary and high school education. In 1985, he enrolled in the Department of Biology at the University of Liaoning. He received a Bachelors of Science honors degree in biology in 1985. Subsequently, he was employed by the Institute of Environment Protection at Shenyang. In 1987, he returned to school and enrolled as a M.S graduate student in the Department of Plant Physiology and Biochemistry in the Division of Basic Science (currently called the College of Biotechnology) at Shenyang Agriculture University. He received a M.S degree in July, 1990. Upon graduation, he was employed by the Division of Basic Science in the College of Traditional Chinese Medicine at Liaoning as a biochemistry lecturer until 1996. Subsequently, he enrolled again as a graduate student in the plant physiology Ph.D. program of the Biology Department at South China Normal University until 1998. In the fall of 2001, he transferred to the Department of Plant Pathology and Crop Physiology at Louisiana State University in Baton Rouge, Louisiana as a graduate student to pursue a Masters degree under the guidance of Dr. M. C. Rush.
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