Apr 2, 1998 - luteolinidin and apigeninidin compounds (Viswanathan et al. 1996) to assess red rot reaction. Recently, colonization offungal infection on cane ...
Indian Journal ofAgricultural Sciences 68 (4) : 226-30, April 1998
Comparison of three testing methods for evaluation of resistance to red rot caused by Colletotrichumfalcatum in sugarcane (Saccharum officinarum) R VISWANATHANi' D MOHANRAF and P PADMANABAW Sugarcane Breeding Institute, Coimbatore, Tamil Nadu 641 007 Received: 17 February 1997
ABSTRACT Red rot, caused by Colletotrichumfalcatum Went is one of the major diseases of sugarcane (S~ccharum officinarum L.) affecting cane stalks. Sugarcane genotypes are tested using both plug and nodal evaluation methods for resistance against red-rot pathogen, before varietal release. These :ecommended testing methods were compared with the newly developed controlled condition testing procedure. Red-rot was evaluated during 1992-93, 1993-94 and 1994-95 seasons under field conditions and controlled condition testing. Disease ratings of the pre-zonal varietal trial clones by 3 different testing procedures showed that the controlled conditions testing had high correlation with plug method compared with the nodal method of evaluation in the field. Testing by controlled condition testing gave more reliable results than field nodal method and many disease escapes noticed in the latter were identified. Plug method ofevaluation also showed less correlation with nodal method. Controlled condition testing was found less injurious and evaluation could be done in a more natural way. Aniong the 3 methods controlled condition testing was suitable to indentifY field tolerant clones with more reliability in a short time.
Key words: Sugarcane, red-rot disease, resistance, plug method, nodal method, controlled condition testing, Colleetrotriehum faleatum, Saeehanml ojficinanlm
Red-rot caused by the fungus Colletotrichumfalcatum Went. (perfect state; Glomerella tucumanensis (Speg.) Arx & Muller) is the major constraint to sugarcane (Sacchanlm ojJicinanim L.) cultivation in India (Agnihotri 1990, Alexander and Viswanathan 1996). Extensive crop damage was reported from 1930's onwards in India and most of the commercial varieties succumbed to the disease. Use of resistant varieties constitute the basis of integrated disease management in sugarcane and these are evolved by crossing proven parents. Sugarcane is highly heterozygous and polyploid in nature and accordingly disease reactions in the progenies vary widely. Several methods have been developed from time to time for screening sugarcane genotypes, germplasm lines and breeding progenies for resistance. Most of the screening techniques developed to screen genotypes are based on disease symptoms on the stalks. In the plug method of inoculation the fungal spore suspension is injected into a bore hole made on the third internode (above ground level) using a cork borer in 5-6 month-old standing stalks (Chona 1954). The disease reaction is graded into,resistant, moderately resistant and susceptible based on the average lesion length. Later, Srinivasan and Bhat (1961) developed a method based on qUalitative characters like drying of tops, lesion width, occurrence and nature of white 1
Scientist,
2, 3
Senior Scientists
spots in the lesion and nodal transgression. Here disease ratings are classified into resistant , moderately resistant, moderately susceptible, susceptible and highly susceptible in a (}-9 scale. A discriminant function for grading sugarcane resistance against red-rot pathogen was reported by Prasada Rao et al. (1978). The method of Sriniv;lSan and Bhat (1961) is being followed by all the sugarcane pathologists to assess disease resistance in sugarcane. Although this method is considered to reveal the physiological tissue resistance it is known to break the natural barriers ofthe host. Hence, nodal methods like IISR method (Singh and Budhraja 1964) and nodal injury methods (Rana and Gupta 1968, Kirtikar et al. 1974) were developed to identifY red-rot resistance without breaking natural barriers of sugarcane stalk. In these methods either the inoculum is applied on the nodal region after removing leaf sheath or placing conidial suspension between leaf sheath and stalk. Disease development in nodal method of inoculation is highly int1uenced by environmental factors (Singh et al. 1983). Recently Mohanraj et al. (1994, 1997) developed a controlled condition testing 1;>ased on screening sugarcane in a temperature and humidity controlled chamber. This method was found rapid, precise and evaluation time was short. Hence, an attempt was made to compare the disease ratings of sugarcane by plug method, nodal method and controlled condition testing method to find out the suitable
April 1998]
COMPARISON OF TIIREE 1ESTING ME1HODS
method among them to assess the disease reaction in a shorter time with more reliable results. MATERIALS AND METHODS
Field trials Field trials were conducted at Padalam, Chengalpattu district of Tamil Nadu during 1992-93, 1993-94 and 199495 crop seasons. Pre-zonal varietal trial clones were planted usually during January-February and pathogen inoculations were carried out during last week of August every year by plug- and nodal-methods. Mixed inoculum from the C. falcatum pathotypes isolated from the infected materials of 'Co 6304', 'COC 671', Co 8001', 'CoC 85061', 'CoC 86062' and 'CoC 92061' were used for disease evaluation. Disease ratings by plug method were scored after 45-60 days irtto resistant, moderately resistant, moderately susceptible, susceptible and highly susceptible as per Srinivasan and Bhat (1961). For nodal method of testing 1 ml of the pathogen inoculum was applied in between sheaths of 3rd and 4th leaves from top using a pasteur pipette. Overhead sprinklers were operated for 15 days from the day of inoculation to maintain high humidity. Here, the disease ratings were categorized into resistant or susceptible after 45 to 60 days (Anonymous 1995). , Controlled condition testing Evaluation by this method was done essentially as per the method developed by Mohanraj et al. (1977). The testing chamber was of3 m x 3 m x 3.6 m dimension and fabricated with steel frames covered with high density polythene sheets. The inside of the chamber was illuminated each day for 8 hr by fluorescent lamps with a total light energy output of320 W (9 w/m 2). A timer controlled humidifier (L&T E-M 1000) was installed to maintain 90% relative humidity inside the chamber. Canes of 8-10 month-stage were brought from the field, the upper 2/3 of the canes with intact leaf sheaths and spindle placed in trays containing wet stand of 10-15 cm height. The lower most node of the stalk was below the surface of the sand bed. Nodes of 6-8 position from the top were selected for inoculation from which leaf sheaths were removed using a fine lmife without injuring the nodal tissues. Pathogen inoculum (2 ml) prepared as for field trials was applied on the selected nodes and covered with thick moist cotton pads and polythene strips. Inoculated canes were incubated under 90% relative humidity and the temperature was maintained at 32°C. After 7-10 days, the inoculated canes were rated as resistant, moderately resistant, moderately susceptible, susceptible and highly susceptible based on the criteria reported earlier (Mohanraj et al. 1997). Red rot disease trials under field conditions and controlled condition testing for 3 seasons to asess their relative merits and other short comings. The data from 3 testing methods were subjected to statistical analysis and correlation matrix was worked out for results of each year.
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REStJLTS AND DISCUSSION
In general, testing by field nodal method gave more number of resistant reactions compared with the other 2 . methods. In 1993, nodal methods of evaluation showed 23 clones as resistant, whereas in plug method it was one and controlled condition testing has no resistant clones. In subsequent years also field nodal method of testing showed many clones as resistant but these clones were found susceptible by plug method and controlled conditions testing method (Table 1). Red-rot severity score was divided into 5 categories, viz resistant, moderately reseistant, moderately susceptible, susceptible and lighly susceptible in plug method and controlled condition testing systems, whereas in tleld nodal method, a clone was rated either as resistant or susceptible. By field nodal method a clone could not be rated for intermediate reactions. When comparison was made between plug method and controlled condition testing there was similarity in their reaction categories. However, some clones rates susceptible or highly susceptible by plug method showed moderate resistance in the controlled condition testing. Statistical analysis in 1993 showed that controlled condition testing and plug method had correlation coefficient of 0.73, controlled condition testing and nodal method 0.28 and plug method and nodal method 0.48. In 1994 controlled condition testing and plug method had correlation coefficient of O. 78, controlled condition testing and nodal method 0.53 and plug method and nodal method 0.61. In 1995, testing the respective figures were 0.64, 0.38 and 0.60. In all the 3 years testing by controlled condition and plug method had higher correlation than the nodal method with plug method or controlled condition testing (Table 1). Serious constraint in the development resistant varieties to red-rot is the frequent breakdown of resistant varieties to the disease as a result of occurence of new races. This has resulted in the removal of the hitherto resistant popular varieties from cultivation (Srinivasan 1962, Padmanaban et al. 1996). At this moment it is imperative to identify clones with durable resistance or clones with field tolerance. Among the methods used to evaluate clones, nodal method came to existence to accurately judge the field-resistance of the clones (Singh and Budhraja 1964, Rana and Gup~a 1968). However, nodal testing results in large number of disease escapes under field conditions. This may be due to lack of favourable parameters under field conditions for disease development. Controlled condition testing was developed to mitigate such shortcomings in the nodal method of testing and to evaluate clones under a near natural conditi~ns ..In the nodal method of testing the inoculum was appbed m between leaf sheaths and this technique gives limited chance for the pathogen to survive and reach the nodal region and initiate infection. However, in the present method the inoculum is applied right on the nodal region known to be the point of entry of the pathogen (Srinivasan and Alexandar 1965) without causing much injury and optimum
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[Vol 68, no. 4
VISWANATIIAN ET AL. Table I Comparison of red rot reactions evaluated by different evaluation techniques 1993
1994
1995
Genotype
Plug method
Nodal method
Controlled condition testing
Genotype
Plug method
Nodal method
Controlled condition testing
Genotype
Plug method
Nodal method
Controlled condition testing
9300 93011 . 93012 93013 93014 93015 93019 93020 93022 93026 93028 93031 93035 93038 93039 93041 93044 93045 93047 93053 93055 93063 93065 93066 93067 93068 93078 93079 93085 CoC671
S 7.0 MR3.0 S 8.0 S 8.0 S 8.0 MS6.0 HS9.0 MS6.0 HS9.0 MR3.0 HS 8.5'~ HS9.0 S 8.0 HS9.0 HS 9.0 MS5.0 H89.0 MS5.0 MSS.O MS5.0 S8.0 HS9.0 R2.0 S 6.0 HS9.0 HS9.0 S 7.0 S 8.0 MR3.0 HS9.0
RI.O RI.O RI.O RI.O RI.O RI.O RI.O RI.O RI.O "RI.O S 8.0 S 8.0 RI.O S 8.0 S 8.0 RI.O RI.O R.1.0 RI.O RI.O S 8.0 RI.O RI.O RI.O S 8.0 S 8.0 RI.O RI.O RI.O S8.0
MS5.0 MS5.0 HS9.0 s 7.0 HS9.0 S 7.0 HS9.0 HS9.0 HS9.0 MR3.0 S6.0 S 7.0 HS9.0 HS9.0 HS9.0 MR4.0 HS8.S MR3.0 MSS.O MS5.0 S 8.0 MR3.0 MR3.0 MS5.0 HS8.5 HS9.0 HS 8.5 HS9.0 MR3.0 HS9.0
94001 94002 94007 94008 94010 94012 94013 94014 94015 94016 94017 94020 94024 94025 94027 94029 94031 94033 94034 94038 94039 94041 94042 94043 CoC671
MR3.0 HS9.0 MR3.5 HS9.0 S 7.5 HS9.0 S 8.0 MR3.5 MS5.4 MS6.0 S.80 HS9.0 MSS.O HS9.0 MSS.2 S 7.5 S 8.0 S 7.5 MSS.O HS9.0 HS9.0 MS5.0 MR3.4 Hs9.0 HS9.0
RI.O S 8.0 RI.O S 8.0 S 8.0 S 8.0 S 8.0 RI.O Rl.O R.1.0 RI.O S8.0 RI.O S 8.0 RI.O RI.O R.1O RI.O RI.O S 8.0 S 8.0 RI.O RI.O S 8.0 S 8.0
MR3.0 HS 8.5 MR3.0 HS9.0 MS5.8 S 8.0 S 8.0 MS4.2 87.2 S 7.5 HS9.0 S 8.0 HS9.0 S 8.9 MSS.O MS5.7 S 8.0 MS6.0 MR3.5 S 8.5 S8.4 MS5.2 MR4.0 S 8.0 HS9.0
95010 950n 95012 95013 95015 95017 95018 95019 95020 95023 95024 95025 95029 95030 95031 95039 95045 95046 95047 95048 95049 95050 95052 95054 95056 95057 95060 95061 95062 95063 95064 95066 95067 95068 95970 95071 95072 95073 95075 95078 95079 950BO 95081 95083 95085 CoC671 COC92061
MS5.8 HS9.0 HS9.0 HS9.0 HS9.0 HS9.0 MS5.8 HS9.0 H89.0 S 8.0 HS9.0 S7.2 MS5.0 HS9.0 HS9.0 HS9.0 HS9.0 HS9.0 HS9.0 HS9.0 HS9.0 HS8.5 HS 8.2 HS9.0 HS8.4 HS8.7 HS8.6 S 7.2 HS 9.0 HS9.0 S 8.0 HS9.0 HS9.0 HS9.0 HS9.0 HS9.0 HS9.0 HS 8.8 HS 8.5 HS9.0 HS 8.2 HSB.I HS9.0 HS9.0 HS9.0 HS9.0 HS9.0
R 1.0 S 8.0 S 8.0 S 8.0 S8.0 S 8.0 RI.O S8.0 88.0 S 8.0 S 8.0 S 8.0 RI.O R. 1.0 S 8.0 S 8.0 S9.0 S 8.0 S 8.0 S 8.0 S 8.0 S 8.0 S 8.0 88.0 S 8.0 .R 1.0 S 8.0 S 8.0 S 8.0 S8.0 R 1.0 S 8.0 S 8.0 S 8.0 RI.O S8.0 S 8.0 S 8.0 S 8.0 S 8.0 S 8.0 SB.O S 8.0 S 8.0 RI.O S 8.0 S 8.0
HS8.5 HS9.0 HS9.0 HS9.0 HS9.0 HS9.0 S 7.5 HS9.0 H89.0 HS8.5 HS9.0 MS6.0 MS5.6 HS9.0 HS9.0 HS9.0 HS9.0 HS9.0 S 8.0 S 7.5 HS 8.5 HS8.8 HS 8.9 HS8.5 HS9.0 HS 9.0 HS9.0 MS6.0 HS9.0 HS9.0 S 8.0 HS9.0 HS9.0 HS9.0 HS9.0 S 8.0 HS9.0 S 8.0 HS 8.5 HS9.0 S 8.0 MS6.0 HS9.0 HS9.0 S B.O HS9.0 HS9.0
Correlation matrix 1.00000
Plug method Nodal 0.43302 method 0.73466 Controlled condition testing Critical value I.Tail (0.05) 2.Tail (0.05)
1.00000 1.00000 0.28544
OJ 119 0.3666
1.00000
1.00000
0.61795
1.00000
0.78926
0.53159
1.00000
0.3302 0.3874
R, Resistant; MR, moderately resistant; S, susceptible, MS, moderately susceptible; HS, highly susceptible
0.60256
1.00000
0.63903
OJ7821
0.2485 0.2934
l.00000
April 1998]
COMPARISON OF THREE TESTING :METHODS
environmental parameters were maintained throughout the testing period. In the nodal method the clones are rated as resistant or susceptible, which are the extremes in the disease reactions. This is another drawback of the method, whereas the present system of evaluation allows determination of intermediate disease reaction categories of moderately resistant, moderately susceptible and susceptible in sugarcane intermediate categories may allow the clones developed to be considered for varietal release since these types were found to have fIeld tolerance/resistance. Both the plug method and controlled condition testing method had high correlation for the disease ratings since the disease evaluation was based on 09 scale developed utilizing different pathogenecity factors of C [alcatum. When reactions of both the methods were compared, plug method showed more susceptibility than the present method since inoculum was applied into the internodal tissues breaking the natural barriers of host resistance. In general for field testing, pathotypes prevailing in a location are used for evaluation and we fail to evaluate the clone with pathotypes of other regions under uniform and identical conditions. Lack of information on the varietial reaction to different pathotypes delays the possibility ofrelease of varieties evaluated in J location in other regions. The present method enables us to take up screening of all genotypes in one place simultaneously against all pathogen races. Another merit is the ability of testing more number of clones in a limited area with more replications and repetitions. Moreover, this system of evaluation allows us to use certain indices for red rot resistanc~ like histopathological parameters (Mohanraj et al. 1994) red rot pigments (Viswanathan et al. 1994) and sugarcane phytoalexins like luteolinidin and apigeninidin compounds (Viswanathan et al. 1996) to assess red rot reaction. Recently, colonization offungal infection on cane tissues were quantifIed based on enzyme linked immunosorbent assay (ELISA) using polyclonal antiserum developed against the fungus. This method was found more convenient to use ELISA and disease reaction of this method are confIrmed again by ELISA (Viswanthan et al. 1997) and histopathological parameters. Presently controlled condition testing method is followed to test large number of parental clones, germpJasm accession, pre-release or ground nursery genotypes and tissue culture derived clones at th.is Institute. It would enable elimination of a large number of susceptible clones at the early stage itself resulting in much savings of time, labour and other resources since many repetitions are possible in an year. Since, the disease reaction of this method is based on different parameters like histological, serological and histochemical besides the actual pathogen reaction, genotypes with true resistance can be picked Up without ambiguities. In many other host pathogen systems also such controlled condition testing involving many pathotypes from
229
dift~rent locations are ~ practice (Pande et al. 1994). Clones identified as resistant by the new method would stand in the field for long time, since the genotypes are passed through a series of confirmatory testing systems. Further, identit1cation of disease resistance in a shorter time in the controlled condition testing would hasten the release of sugarcane genotypes for commercial cultivation. ACKNOWLEDGMENT We are thankful to Mis Madhuranathakam Co-op Sugar Mills Ltd, Padalam for providing fIeld facilities. REFERENCES Agnihotri V P. 1990. Diseaes ofSugarcane and Sugarbeet, 483pp. Oxford & IBH PublishIDg Co, New Delhi. Alexandar K C and Viswanathan R. 1996. MfUor diseases afIecting sugarcane production in India and recent experiences in quarantine. (in) Sugarcane Germplasm Conservation and Exchange, Croft B J, Piggin C M, Wallis E S and Hogarth D M (Eds). Proceedings 67, Australian Centrefor International Agricultural Research, Canberra, Australia, pp 46-8. Anonymous, 1995. Annual Report, All India Coordinated Sugarcane Improvement Project, Plant Pathology, Indian Institute of Sugarcane Research, Lucknow, pp 21-':3. Chona B L. 1954. Relative resistance of sugarcane varieties to red rot. Indian Journal ofAgricultural Sciences 24: 301-15. Kirtikar, Gupta S C and KureeI D G. 1974. Screening of varieties for red rot resistance. Proceedings, 15th Congress of International Soceity of Sugarcane Technologists 1 : 189-93. Mohanraj D, Padmanaban P, Viswanatban R and Alexander K C. 1994. Rapid evaluation of resistance against red rot disease of sugarcane under controlled conditions. (in) Proceedings ofNational Symposium on Ultrastructure, Cytology, Sexuality and Variability in Plant Pathogens held during 24-27 January 1994 by Indian Phytopathological Society at Tamil Nadu
Agricultural University, Coimbatore, pp 8-9. D, Padmanaban P, Viswanathan R and Alexander K C. 1997. Sugarcane screening for red rot resistance. Sugarcane 3: 18-23. Pande S, Thakur RP, Karunakar R I, Bandyopadhyay R and Reddy B V S. 1994. Development of screening methods and identification of stable resistance to anthracnose in sorghum. Field Crops Research 38: 157-66. Paclmanaban P, Mohanraj D, Viswanathan R, Rao M M, Prakasam N, Jothi R and Alexandar K C. 1996. Differential interaction of clones to patbotypes of Colletotrichum falcatum Went. Sugarcane 4 : 16--20. Prasada Rao K K, Sarma M N, Satyanarayana V and Atchutarama Rao ·M. 1978. Discriminate function as a reliable guide for assessing varietal reaction to red rot of sugarcane. Proceed-
Mohanr~
ings, 16th Congress of International Society of Sugarcane . Technologists 1 : 395-400. Rana 0 S and Gupta S C. 1968. An easy method of screening out
red rot susceptible varieties at initial stage of multiplication. Indian Sugar 18 : 447-52.
Singh K and Budhraja TR. 1964. Method of inoculating sugarcane for red rot. Plant Disease Reporter 48: 191-3. Singh R P, La1 S and Singh K. 1983. Development of donnant
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nodal infections of Colletotrichum falcatum Went in sugarcane. Proceedings, Annual Convention of Sugarcane Technologists Association ofIndia 47: 79-90. Srinivasan K V. 1962. Some observations on variations in the red rot pathogen Glomerella tucumanensis (Speg.) Arx & Muller. Proceedings, International Society of Sugarcane Technologists 1.1 th Congress. 1 : 795-802. Srinivasan K V and Alexander K C 1965. A study of mode of nodal infection and occurrence of dormant infection of red rot. Proceedings, All India Conference ofSugarcane Research and Development Workers 5 : 676-84. Srinivasan K V and Bhat N. 1961. Red rot of sugarcane-
