PHOTOACOUSTIC SPECTROSCOPY FOR IDENTIFICATION AND DIFFERENTIAL DIAGNOSIS OF T. INDICA WITH OTHER SEED-BORNE PATHOGENS OF WHEAT AND RICE

This article was downloaded by: [Tufts University] On: 22 October 2014, At: 07:08 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Instrumentation Science & Technology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/list20 PHOTOACOUSTIC SPECTROSCOPY FOR IDENTIFICATION AND DIFFERENTIAL DIAGNOSIS OF T. INDICA WITH OTHER SEED-BORNE PATHOGENS OF WHEAT AND RICE Vikrant Gupta a , Anil Kumar b , G. K. Garg a & A. K. Rai c a Department of Molecular Biology and Genetic Engineering , G. B. Pant University of Agriculture and Technology , Pantnagar, UP, 263 145, India b Department of Molecular Biology and Genetic Engineering , G. B. Pant University of Agriculture and Technology , Pantnagar, UP, 263 145, India c Department of Physics , G. B. Pant University of Agriculture and Technology , Pantnagar, UP, 263 145, India Published online: 20 Aug 2006. To cite this article: Vikrant Gupta , Anil Kumar , G. K. Garg & A. K. Rai (2001) PHOTOACOUSTIC SPECTROSCOPY FOR IDENTIFICATION AND DIFFERENTIAL DIAGNOSIS OF T. INDICA WITH OTHER SEED-BORNE PATHOGENS OF WHEAT AND RICE, Instrumentation Science & Technology, 29:4, 283-293, DOI: 10.1081/CI-100105717 To link to this article: http://dx.doi.org/10.1081/CI-100105717 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. 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Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions http://www.tandfonline.com/loi/list20 http://www.tandfonline.com/action/showCitFormats?doi=10.1081/CI-100105717 http://dx.doi.org/10.1081/CI-100105717 http://www.tandfonline.com/page/terms-and-conditions http://www.tandfonline.com/page/terms-and-conditions PHOTOACOUSTIC SPECTROSCOPY FOR IDENTIFICATION AND DIFFERENTIAL DIAGNOSIS OF T. INDICA WITH OTHER SEED- BORNE PATHOGENS OF WHEAT AND RICE Vikrant Gupta,1 Anil Kumar,1,* G. K. Garg,1 and A. K. Rai2 1 Department of Molecular Biology and Genetic Engineering and 2 Department of Physics, G. B. Pant University of Agriculture and Technology, Pantnagar 263 145 (UP), India ABSTRACT The photoacoustic study of dry spores (PAS) described in the present paper proved to be a suitable technique (when compared with other conventional methods) for the identification of Karnal bunt (KB) amongst other seed borne pathogens. Spores of six pathogens, i.e., Tilletia. indica, T. barclayana, Ustilago tritici, Ustilaginoidea virens, Helminthosporium sativum, and Alternaria triticina were isolated and their PA spectra recorded in the wave- length range of 200 to 800 nm and at a modulation frequency of 18 Hz using an indigenous photoacoustic spectrometer. The number and the intensities of peaks in the PA spectra obtained from pathogens were compared. Our results revealed that, for some diseases, a few extra peaks were observed. Likewise, for some of the diseases, common peaks were observed, but of different intensities. Apart from these, some addi- tional peaks, characteristic for particular pathogens, were also observed. In PA spectroscopy, the characteristic peak patterns and signal strengths consti- tute a basis for differential diagnosis of the disease. Our results clearly demonstrated that various biomolecules are present in varying proportions in the dry spores of the six pathogens. INSTRUMENTATION SCIENCE & TECHNOLOGY, 29(4), 283–293 (2001) 283 Copyright © 2001 by Marcel Dekker, Inc. www.dekker.com *Corresponding author. D ow nl oa de d by [ T uf ts U ni ve rs ity ] at 0 7: 09 2 2 O ct ob er 2 01 4 INTRODUCTION Wheat (Triticum aestivum L.) is one of the major food crops of the world. India ranks second in area and production of wheat in the world, and is now capable of exporting it to other countries. Prevalence of different diseases in wheat growing areas has been recorded; these have caused appreciable losses in certain years. Karnal bunt (KB) disease of wheat, caused by the fungus Tilletia indica (Syn Neovossia indica), is a very important disease in the northwestern regions, particularly Uttar Pradesh, Himachal Pradesh, Jammu, and Kashmir and Punjab states of India. Karnal bunt, was first detected at Karnal (Haryana) in India by Mitra.(1) Since then, the disease has spread to northwestern states of India, parts of Bihar, West Bengal, northern parts of Madhya Pradesh, Rajasthan, and Junagarh district of Gujarat.(2,3) Outside India, the disease has been reported to occur in Pakistan, Iraq, Afganisthan, and Nepal.(4,6). It was detected in Mexico in 1970(5) and in southern parts of the United States in 1996.(7) Normally, it appears sporadically in iso- lated areas as a minor disease, causing insignificant loss in yield, but in certain years, it assumes epiphytotic proportions.(8,10). The disease affects common bread wheat and, to a lesser extent, durum wheat, triticale, and related species, significantly reducing the grain quality.(5,11) For successful management of KB, it is essential to check its further spread to disease-free areas through the use of disease-free seeds. Hence, there is a strong need towards developing suitable and rapid biophysical techniques for detection of KB pathogen in wheat lots. Many techniques can be employed to detect KB or to differentiate it from other pathogens. Conventional techniques include light microscopy based teliospore detection and morphological studies.(12) Teliospores of the common bunt pathogen of wheat (Tilletia caries) and the dwarf bunt fungus (T. contro- versa) are difficult to distinguish by light and electron microscopy. With the emerging modern diagnostic tools, efforts are on, with limited success, to develop specific immunological and DNA based probes to diagnose the KB disease.(13,14) It is well established that differentiation of fungal pathogen was dependent upon the qualitative and quantitative differences of specific markers present at the surfaces of pathogens. The number of these markers is very small compared to common structural components of two fungal pathogens. It, therefore, becomes extremely difficult to pinpoint these markers in fungal spores/teliospores by conventional chemical and/or biochemical methods. Recently, we have demonstrated that PAS may be successfully used to moni- tor and analyse different diseases in plants.(15,18) When using PAS, there is no need for sample preparation; the PA spectra of dry spores may be directly recorded by scanning the wavelength of exciting radiation. Different diseases may be identified by the characteristics peaks (bands) expected to be obtained due to synthesis of different biomolecules. In the present investigation, PAS of dry spores was employed to differentiate Tilletia indica from other seed borne pathogens. 284 GUPTA ET AL. D ow nl oa de d by [ T uf ts U ni ve rs ity ] at 0 7: 09 2 2 O ct ob er 2 01 4 EXPERIMENTAL Six seed borne pathogens of wheat and rice used for the present study were (a) Tilletia indica, (b) T. barclayana, (c) Ustilago tritici, (d) Ustilaginoidea virens, (e) Helminthosporium sativum, and (f) Alternaria triticina. Spores of Ustilaginoidea virens (false smut) and T. barclayana were extracted from the seeds of infected rice with the help of needle, forceps, and surgical blades. The seeds were incised with the surgical blade and the spore mass loosened with the help of a needle. The spores were released on shaking the incised seeds in closed glass vials. By simple sieving, the debris was separated from the spores. Similarly, teliospores of T. indica and spores of Ustilago tritici were extracted from the infected wheat seeds of cultivar HD 2328. Cultures of Helmintoshporium sativum and Alternaria triticina were grown and spores were taken after sporulation. Differentiation of fungal spores/teliospores of various pathogens was based on characteristic absorption bands. The physics of the PA effect involves conver- sion of photon energy into an acoustic signal. This occurs when incident radiation is periodically interrupted (at audio frequencies). The PA spectrum of each pathogen was recorded using a single beam PA spectrometer.(17) A 300 watt xenon high pressure lamp (Oriel Corporation 66083) serves as an excitation source. Before entering the monochromator, the white light was modulated by a mechani- cal chopper (Stanford SR 540). A monochromator (CEL, India, HM 104) with 1200 grooves/mm grating blazed at 500 nm was used to disperse light; spectral dis- persion was 3.3 nm/mm. All spectra were recorded between 200 nm and 800 nm. The modulated radiation (18 Hz) was focused into the sample compartment of an indigenous PA cell (2.0 cm diameter, 2 mm depth), and provided with a sen- sitive gas microphone (Electret microphone). The PA signal from the cell was amplified and fed to the lock-in amplifier (Stanford SR 530) for synchronous phase sensitive detection. A time constant of 30 sec was used for all measure- ments. The spectra were normalized by ratioing (at each wavelength) the PA signal acquired from the sample with that obtained from a carbon black. Two selected procedures were used for extraction of teliospores/spores surface components of the six pathogens. Water and HCl extracts of pathogens (2 mg/mL) were prepared and subjected to spectrophotometric scanning in the range of 190–400 nm. RESULTS The PA spectra of pathogen spores, i.e., T. indica, (Karnal bunt), T. barclayana, (Ricebunt), Ustilaginoidea virens, (False smut), Ustilago tritici (Loose smut), Alter- naria triticina, and Helminthosporium sativum (Helminthosporium) were recorded by loading the pathogen spores directly into the PA cell, and are shown in Figures 1 & 2. PHOTOACOUSTIC SPECTROSCOPY 285 D ow nl oa de d by [ T uf ts U ni ve rs ity ] at 0 7: 09 2 2 O ct ob er 2 01 4 286 GUPTA ET AL. F ig u re 1 . P ho to ac ou st ic s pe ct ra o f te li os po re s/ sp or es o f w he at a nd r ic e pa th og en s. D ow nl oa de d by [ T uf ts U ni ve rs ity ] at 0 7: 09 2 2 O ct ob er 2 01 4 PHOTOACOUSTIC SPECTROSCOPY 287 F ig u re 2 . P ho to ac ou st ic s pe ct ra o f sp or es o f w he at a nd r ic e pa th og en s. D ow nl oa de d by [ T uf ts U ni ve rs ity ] at 0 7: 09 2 2 O ct ob er 2 01 4 Spores of T. indica (Karnal bunt) exhibit a single, very strong peak at 272 nm and weak PA signals at wavelengths 292 nm and 232 nm, respectively (Fig. 1). Likewise, T. barclayana (Rice bunt) also showed two bands, one (strong) at 292 nm and another broad and weak band at 232 nm. In addition to those bands, very weak bands at 372, 552, and 652 nm (Table 1, Fig. 1) were obtained. In contrast to T. indica, U. virens displayed a strong band (at 232 nm), a less intense band (at 292 nm), and very weak bands at 392 and 592 nm. (Table 1, Fig. 2). The PA spectra of Ustilago tritici showed a strong band at 232 nm and a new weak band at 272 nm. However, a band at 292 nm was not observed from this pathogen (Fig. 2). The PA spectrum of Alternaria triticina showed a strong band at 292 nm and a band of medium intensity at 232 nm . In addition to these two bands (as observed in other pathogens), five weak bands were also observed at 432, 572, 612, 692, and 752 nm in this pathogen (Table 1, Fig. 1). The PA spectrum of Helminthosporium sativum exhibited trends (very strong band at292 nm) that are very different from those observed with other pathogens used in the present study. In addition to these, bands of medium intensity were observed at 232 nm, 412 nm, and 752 nm. It also showed two weak bands at 352 and 492 nm (Table 1, Fig. 2). Figure 3 compares the bands obtained for different pathogens. 288 GUPTA ET AL. Table 1. Determination of Various Bands in Photoacoustic Spectra of Spores of Pathogens Photoacoustic Spectra Pathogens Wavelength (nm) Photoacoustic Signal Tilletia indica 232 0.70 292 3.72 Tilletia barclayana 232 0.92 292 2.30 372 0.39 552 0.33 652 0.37 Ustilaginoidea virens 232 2.34 292 0.75 Ustilago tritici 232 1.73 272 0.90 Alternaria triticina 232 1.45 292 2.58 432 0.43 572 0.37 612 0.35 692 0.42 752 0.37 Helminthosporium sativum 232 1.37 292 3.53 352 0.44 412 0.98 492 0.31 752 0.76 D ow nl oa de d by [ T uf ts U ni ve rs ity ] at 0 7: 09 2 2 O ct ob er 2 01 4 PHOTOACOUSTIC SPECTROSCOPY 289 Figure 3. Diagram representing various peaks in photoacoustic scans of the pathogens. D ow nl oa de d by [ T uf ts U ni ve rs ity ] at 0 7: 09 2 2 O ct ob er 2 01 4 DISCUSSION In plant pathology, the PAS technique is gaining importance for obtaining valuable information about various diseases in plants by comparing PA spectra of normal and diseased plants.(16,17) In plant sciences, PA spectroscopy is mainly applied to studies of photosynthesis.(18) Greene et al.(19) applied the PA spec- troscopy technique in the study of fungal contamination in corn. Unique bands in the PA spectra are characteristic of a particular disease, and can be employed for the diagnosis of diseases, even at the seed level. In the present investigation, an attempt was made to differentiate teliospores of Karnal bunt with other bunt, smut, and related seed borne pathogens. The PA spectra of dry spores of the pathogens were recorded in the 200-750 nm region at a modulation frequency of 18 Hz and the number of observed peaks and their intensities were compared. The band at 232 nm was common to PA spec- tra acquired from of all the pathogens used in the present study. However, the intensity of the band varied from pathogen to pathogen, showing the following relationship concerning the intensity: U. virens > U. tritici > A. triticina > H.sativum>, T. barelayana > T. indica. These observations showed that a compound (biomolecule) having an absorption band at 232 nm (probably amino acids) is common to all pathogens, but its amount varies according to the observed intensity pattern.. With exception of Ustilago tritici, all pathogens showed a very strong signal at 292 nm. This clearly showed that the concentration of biomolelcules having an absorption band at 292 nm is very large in comparison to other biomolelcules present in the pathogens. The concentration of this molecule (having absorption band at 292 nm) follows the trend : T. indica > Helminthosporium > Alternaria triticina > T. barclayana > U. virens. The spectrum of Ustilago tritici showed absence of molecules absorbing at 232 nm that was found even in large concentration in the five other pathogens. However, the presence of new biomolecules absorbing at 272 nm was detected. The band at 232 nm in the PA spectra of T. barclayana is broad in comparison to those observed from the remaining other five pathogens, which suggests presence of different functional moities in the molecule of T. barclayana enabling discrimi- nation from other pathogens. These can be confirmed by subsequent biochemi- cal/Raman and infrared spectroscopy. The results revealed that, based on intensities of bands at 292 nm and 232 nm, one can clearly distinguish the above pathogens, which is otherwise not possi- ble when using conventional methods (Fig. 4 of absorption spectra). The intensity ratios of the bands (Intensity of bands at 292/Intensity of bands at 232 nm) in pathogens T. indica, T. barclayana, U. virens, U. tritici. A. triticina, and Helminth- osporium sativum is 5.3, 2.5, 0.3, 0.0 (zero), 1.8, and 2.8, respectively. This result showed that T. indica, U; virens, and U. tritici are easily identified by high, very low, and zero intensity ratios, respectively. 290 GUPTA ET AL. D ow nl oa de d by [ T uf ts U ni ve rs ity ] at 0 7: 09 2 2 O ct ob er 2 01 4 The intensity ratio of T. indica is twice that of T. barclayana. It is difficult to differentiate between T. barclayana and Helminthosporium on the basis of this intensity ratio, as the latter is nearly the same for these two pathogens. But the Helminthosporium may be identified from other pathogens by its well defined characteristic bands in the visible region, i.e., at 352, 412, and 752 nm.. The pres- PHOTOACOUSTIC SPECTROSCOPY 291 Figure 4. Overlay of spectrophotometric scans of extracts of spores of pathogens. A. Alternaria triticina; F. Ustilagenoidea virens; H. Helminthosporium sativum; K. Tilletia indica; R. Barclayana; U. Ustilago tritici. (A) Aqueous extract. (B) HCl extract. D ow nl oa de d by [ T uf ts U ni ve rs ity ] at 0 7: 09 2 2 O ct ob er 2 01 4 ence of a small number of bands in the visible region, whereas the presence of only two bands in UV region in U. virens and U. tritici indicates some resem- blance as far as molecular configurations of these pathogens are concerned. When a comparison was made between Helminthosporium sativum and Alternaria triticina, both seed borne pathogens of wheat, the PA spectrum of the former showed unique, well defined bands in the visible range, i.e., at 352, 412, and 752 nm. These were clearly not observed with other investigated pathogens . Unlike PA spectroscopy, conventional absorption spectroscopy is unable to make a differential diagnosis of seed borne pathogens. As observed in Fig. 4, the absorption spectra of these pathogens showed nearly similar band patterns with the intensities of the bands being almost equal. Additionally, being a destructive technique, the amount of spore sample required for analysis by conventional absorption spectroscopy is higher (i.e., preparation of aqueous sonicated extracts of spores). PAS is a non-destructive technique and requres no special sample preparation. Our results showed that PA spectroscopy is a suitable non-destructive tech- nique for distinguishing pathogens of different genera and species. This technique proved useful for differential diagnosis of various seed borne pathogens of wheat and rice. Since testing of the seed health condition is an integral component of any combative strategy for disease management, further studies are in progress to diagnose seed borne disease at the seed level. Eventually, PA might be used as a rapid, sensitive, and economical detection method in seed certification standards and plant quarantine regulations. Presently, PA spectroscopy is tedious to perform and time consuming due to manual setting of equipment and its indigenous design. Attempts are being made to reduce the time taken in experiments by pro- viding automation and computation capabilities. ACKNOWLEDGMENT Resources from a project funded by the Indian Council of Agricultural (F. No. 3-23/97. PP), New Delhi, to the author (AK) has been used for preparation of this manuscript. REFERENCES 1. Mitra, M. A New Bunt on Wheat in India. Ann. Appl. Biol. 1931, 18, 178-179. 2. Singh, A.; Prasad, R. Control of Karnal Bunt of Wheat by a Spray of Fungicides. Indian J. Mycol. Plant Pathol. 1980, 10, 1. 3. Gill, K.S.; Sharma, I.; Aujla, S.S. Karnal Bunt and Wheat Production. A Report; Punjab Agricultural University: Ludhiana, 1993. 4. Joshi, L.M.; Singh, D.V.; Srivastava, K.D.; Wilcoxon, R.D. Karnal Bunt: A Minor Disease That is Now in Threat to Wheat. Bot. Rev. 1983,49, 309-330. 292 GUPTA ET AL. 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Study of Loosesmut Disease in Wheat Plant with Photoacquistic Spectroscopy. Proc. Nat’l. Sympos. Recent Adv. Laser Molec. Spectros. DDU 1998, Gorkhpur Univ., P-44, 64. 17. Joshi, S.; Rai, A.K. Fabrication and Performance of Photoacoustic Cell for the Studies of Molelcules of Biological and Agricultural Importance. Asian J. Phys. 1995, 4, 265- 282. 18. Buschmann, C. Photoaccoustic Spectroscopy and its Application in Plant Science. Botan. Acta 1990, 103, 9-14. 19. Greene, R.V.; Gordon, S.H.; Jackson, M.A.; Bennett, G.A.; McClelland, J.F.; Jones, R.W. Detection of Fungal Contamination in Corn: Potential of FTIR-PAS and DRS. J. Agric. Food Chem. 1992, 40, 1144-1149. Received March 31, 2000 Manuscript 1239 Accepted September 27, 2000 PHOTOACOUSTIC SPECTROSCOPY 293 D ow nl oa de d by [ T uf ts U ni ve rs ity ] at 0 7: 09 2 2 O ct ob er 2 01 4