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Farrer Centre, School of Agricultural and Veterinary Studies, Charles Sturt University, P.O. Box 588, Wagga Wagga, NSW 2678, Australia
E.J. Cother
N.J. Cother
NSW Agriculture, Agricultural Institute, Forest Road, Orange, NSW 2800, Australia
G.J. Ash
J.D.I. Harper
Farrer Centre, School of Agricultural and Veterinary Studies, Charles Sturt University, P.O. Box 588, Wagga Wagga, NSW 2678, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Two fatty acid analysis protocols (the MIDI and a modified MIDI method) were investigated for their utility to characterize and differentiate Rhizoctonia oryzae and R. oryzae-sativae isolates from four countries. Only the modified MIDI method permitted a clear differentiation between the two species, regardless of the isolates’ country of origin. The modified MIDI method gave the most consistent and reproducible fatty acid results. The failure of the MIDI method to differentiate between R. oryzae and R. oryzae-sativae isolates suggests that the 30 minutes saponification step is insufficient to completely break the cell wall of these two species. This study demonstrated that fatty acid profiles, obtained by the modified MIDI protocol, have the potential as a diagnostic tool for both R. oryzae and R. oryzae-sativae.
Key words: FAME, fungi, identification, MIDI
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The recent discovery of both pathogens highlighted the need for rapid and reliable techniques for the identification of Rhizoctonia spp. isolated from diseased tissue.
Numerous identification techniques have been developed to identify Rhizoctonia spp., including morphological examination (Duggar 1915, Butler and Bracker 1970) and nuclear staining (Parmeter and Whitney 1970), anastomosis testing (Parmeter et al 1969), pectic zymogram testing (Sweetingham et al 1986, Neate et al 1988) and various molecular techniques ( Jabaji-Hare et al 1990, Johanson et al 1998).
Total cellular fatty acids analysis is one of the latest methods used for Rhizoctonia identification (Stevens Johnk and Jones 1992). Cellular fatty acid composition is a reliable and accurate technique commonly used to identify bacteria and yeasts (Stahl and Klug 1996, Larkin and Groves 2003). A common protocol used for this identification technique is the MIDI method (Microbial Identification System, Microbial ID Inc., Newark, Delaware) which has been shown to give highly reproducible results for fungi (Miller and Berger 1985, Gudmestad et al 1988). By slightly modifying the protocol used in the MIDI method, Stevens Johnk and Jones (1992, 1993, 1994, 2001) demonstrated that this new technique could be used to differentiate between R. solani anastomosis groups as well as between subgroups within anastomosis groups AG 1, AG 2–2, AG 3 and AG 4. The method also was used successfully with other fungal genera (Lopes da Silva et al 1998, Larkin and Groves 2003).
In 1996 Matsumoto et al described a new total cellular fatty acid analysis protocol and successfully differentiated between isolates of R. solani AG 1-IA, AG 2–2-IIIB, R. oryzae, R. oryzae-sativae and R. fumigata. Priyatmojo et al (2002) used a similar protocol to differentiate between isolates of R. circinata, R. oryzae and R. zeae.
This aim of this study was to evaluate and compare the MIDI method and a modified version as discriminatory tools for R. oryzae and R. oryzae-sativae.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). All isolates were subcultured on one-quarter-strength potato-dextrose agar (PDA) and incubated at 30 C. Plates were regularly inspected for 2 wk to confirm isolate identification and culture purity. Four Uruguyan isolates, originally labeled as R. oryzae-sativae, presented slightly different morphological characteristics compared with referenced R. oryzae-sativae isolates. Therefore as a precaution all Uruguayan isolates were submitted to anastomosis testing to confirm identification. R. oryzae-sativae and R. oryzae belong to the AG-Bb and WAG-O anastomosis group respectively. The anastomosis tester isolate for AG-Bb is the Japanese isolate C-455 (ATCC 76135). The tester for WAG-O is the Japanese isolate C-521 (ATCC 76154). Both testers can be ordered from the American Type Culture Collection, 12301 Parklawn, Rockville, Maryland 20852 (Sneh et al 1991). The five Uruguayan isolates were tested with tester C-455 using the protocol developed by Parmeter et al (1969). The anastomosis testing was repeated twice for each isolate tested.
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Two methods were used. The protocol of Miller and Berger (1985), hereafter referred to as the MIDI method, was used as a standard technique. Matsumoto (1996) used a method of Gudmestad et al (1988) "with slight modifications". Although it has little resemblance to that method, its essential difference is a 3 h saponification step compared with the 30 min step in the MIDI protocol. The second method used was a modified MIDI method employing 3 h saponification, with the remainder of the normal protocol followed.
Fatty acid methyl esters (FAME) were analysed using a Hewlett-Packard 6890 gas chromatograph with a J&W Ultra 2 capillary column and flame ionisation detector. Hydrogen was used as the carrier gas. Fatty acids were identified by their retention time using the peak naming table in the MIDI software (MIDI Inc., Newark, Delaware; version 4.5). The mean of the fatty acid composition of five separate extractions of lyophilised mycelium was calculated for statistical analysis.
Data analysis.— – Data were subjected to an analysis of variance (ANOVA). Variability in individual fatty acid composition with respect to both individual and overall composition among isolates was analyzed by the Walter-Duncan K-ratio t test (K = 100, t = 0.05) using Genstat 5.0. Canonical variate analysis (Genstat 5.0) was used to test differences among R. oryzae, R. oryzae-sativae and the four Rhizoctonia sp. isolates from Uruguay. Relatedness among the isolates based on fatty acid composition was assessed with cluster analysis (nearest neighbor method, Euclidean distance) using STATGRAPHICS Plus 5.1 between R. oryzae and R. oryzae-sativae isolates.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Total cellular fatty acid analysis.— –
MIDI method..
Ten fatty acids were detected in the 30 isolates tested when using the MIDI method (TABLE II). Significant differences (P < 0.05) in fatty acid composition were observed among both Rhizoctonia spp. for five of the 10 fatty acids detected when using this method. The column configuration did not permit differentiation of the closely related acids 16:1 
7c and 15 iso 2OH, and between 18:0 anteiso and 18:2 6,9c. FAME with similar retention times are grouped as Summed Feature 3 and 5, respectively. In both Rhizoctonia spp., Summed Feature 5, 16:0 and 18:1 9c fatty acids accounted for >90% of each fatty acid profile. Although Summed Feature 5 was the most predominant fatty acid in both species, quantitative differences were observed. The percentage of Summed Feature 5 was significantly higher in the R. oryzae isolates (73.1%) than in the R. or yzae-sativae isolates (56.1%).
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). The dendogram resulting from average linkage cluster analysis showed that the clustering of R. oryzae and R. oryzae-sativae isolates was unrelated to species, geographic origin or host (FIG. 2).
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) and significant differences (P < 0.05) in fatty acid composition were observed among both Rhizoctonia spp. for seven out of 11 fatty acids detected. As observed with the MIDI method, Summed Feature 5 (18:0 anteiso/18:2 6,9c), 16:0 and 18:1 9c were the predominant fatty acids and accounted for >80% of each fatty acid profile. Summed Feature 5 was again the predominant fatty acid in both species, and quantitative differences were observed. The percentage of Summed Feature 5 was significantly (P < 0.05) higher in the R. oryzae isolates (77.77%) than in the R. oryzae-sativae isolates (42.99%).
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). R. oryzae and R. oryzae-sativae isolates formed two distinct clusters, each clustering within a Euclidean distance of 2.66 and 1.72 respectively (FIG. 4). Isolates representing R. oryzae and R. oryzae-sativae were clustered with a distance of 4.26 (FIG. 4). The grouping of R. oryzae and R. oryzae-sativae isolates was not related to geographic origin or host (FIG. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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showed that the method of Miller and Berger (1985) (essentially the MIDI method) gave good recovery of fungal fatty acids, gave highly repeatable results and was easy to use. In addition it is probably the most common fatty acid method for characterization of fungi and bacteria. However, the analysis of total cellular fatty acids from Rhizoctonia spp. using the MIDI method did not permit differentiation between isolates of R. oryzae and R. oryzae-sativae. However a clear differentiation between species, regardless of country of origin, was obtained when using the modified method. Furthermore, when the methods were compared, significant differences (P < 0.05) in fatty acid composition were observed for both R. oryzae and R. oryzae-sativae. The modified MIDI method gave the most consistent and reproducible fatty acid results. The extraction of fatty acids was more consistent between samples of the same isolate when the modified method was used. For example, the amount of 15:0 in R. oryzae-sativae isolate DAR 76459 ranged from 0.91 to 2.18 between five replicate samples using the normal MIDI protocol but only varied from 1.32 to 1.66 when extracted by the modified method.
Müller et al (1994) noted that fungal cell walls were resistant and maintained their original shape through the extraction procedure. They were able to increase the extraction of fatty acids (using the method of Miller and Berger 1985) by disruption of cells walls through homogenizing the samples in a ball mill. The essential difference between the MIDI method and the modified MIDI method is the saponification step, which is 2.5 h longer for the modified MIDI method. In both protocols the saponification is initiated to break the bacterial/fungal cell wall to expose the phospholipid bilayer membrane. Thirty min saponification is a standard step for bacterial fatty acid extraction (Gudmestad et al 1988) and has been used successfully to differentiate and characterize species and subspecific groups of Penicillium (Lopes da Silva et al 1998), Rhizoctonia (Steven Johnk and Jones 1992, 1993, 1994, 2001) and Phytophthora (Larkin and Groves 2003). However previous studies on the fatty acid composition of R. oryzae and R. oryzae-sativae always have been done using a longer saponification step (Matsumoto et al 1997, Priyatmojo et al 2002, Matsumoto 2001). Our results suggest that a 30 min saponification step falls short of breaking R. oryzae and R. oryzae-sativae cell walls to expose the entire phospholipid bilayer membrane. This may be achieved by prior homogenization of the sample.
Our results differ from the limited published data for R. oryzae and R. oryzae-sativae. Matsumoto et al (1997) recorded levels of 14:0 and 16:0 fatty acids in R. oryzae-sativae similar to those recorded in this study but considerably different levels of 15:0, 18:0 and 18:1 9c measured by either of the methods we used. There was little similarity between the levels of individual fatty acids of R. oryzae reported by Matsumoto et al (1997), Matsumoto (2001) or by Priyatmojo et al (2002) and those measured in this study.
Matsumoto et al (1997) demonstrated that their fatty acid analysis method could be used to differentiate Japanese isolates of R. oryzae and R. oryzae-sativae. Our study suggests that fatty acid profiles, obtained by the modified MIDI protocol, have the potential to be used as a diagnostic tool for both R. oryzae and R. oryzae-sativae regardless of isolate origin. DAR 76484 was the only Uruguayan isolate to anastomose successfully with tester C-455. As suspected before testing, isolates 6 rice, 1 soil, 2 soil and 3 soil indeed had been identified inaccurately as R. oryzae-sativae.
Leaf sheath diseases of rice are relatively difficult to diagnose due to the similarity of the symptoms (Matsumoto et al 1997, Matsumoto 2001). Early symptoms of sheath spot (caused by R. oryzae) in particular easily can be confused with aggregate sheath spot (caused by R. oryzae-sativae) symptoms, and it often is necessary to isolate and subculture the pathogen for formal identification. This study showed that fatty acid analysis can be used successfully to differentiate among R. oryzae and R. oryzae-sativae isolates regardless of their country of origin. The fatty acid analysis showed that four Uruguyan isolates were mislabeled as R. oryzae-sativae (data not shown), thus underlining the ability of the fatty acid analysis to differentiate between species that morphologically are relatively similar and hard to identify and demonstrating the usefulness of this technique.
Culture conditions are known to influence fatty acid composition in bacteria and fungi, and culture age and temperature are critical parameters. The common growth period for fungi is 4 d (Priyatmojo et al 2002, Steven Johnk and Jones 1993), but Pankhurst et al (2001) and Matsumoto et al (1997) found that 7 d was preferred for adequate hyphal growth and recovering the most fatty acids. We chose 7 d because some isolates of R. oryzae-sativae grew in a jelly-like consistency and insufficient mycelium was produced after 4 d. It is likely that the optimum growth period will need to be established for each fungal genera and that the period might be isolate-specific. Uniformity of procedure is the critical issue. Modern laboratories can achieve the standardization of cultural conditions and extraction protocols required for the successful application of fatty acid analyses to bacteria. Adoption of a uniform practice for fungal growth and extraction would allow the comparison of fungal data between laboratories. Nevertheless, in the absence of such uniformity, fatty acid analysis is a useful discriminatory tool for fungal identification within individual laboratories.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: vlanoiselet{at}csu.edu.au
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LITERATURE CITED |
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