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Pak. J. Bot., 40(1): 415-420, 2008.


  SELECTION OF IN VITRO TECHNIQUE FOR PATHOGENICITY
      AND SCREENING OF WHEAT CULTIVARS AGAINST
                 BIPOLARIS SOROKINIANA
  SHAMIM IFTIKHAR, SHAHZAD ASAD, ANJUM MUNIR AND IFTIKHAR AHMAD*

   Crop Diseases Research Program, Institute of Plant and Environmental Protection,
    National Agricultural Research Centre, Park Road, Islamabad-45500, Pakistan
             *Member Plant Sciences, PARC, P.O.Box, 1031, Islamabad

                                             Abstract

     Bipolaris sorokiniana (Cochliobolus sativus) is a seed and soil borne pathogen causing leaf
blight or spot blotch of wheat worldwide. In Pakistan the pathogen was identified as predominant
leaf spot causing fungus in wheat growing areas during 2004 – 2006. A lab based technique has
been standardized to study the pathogenic nature of the pathogen, its variability/ aggressiveness and
preliminary screening of cultivars/ germplasm before going to explore the sources of resistance
under natural conditions. The purpose of this study was to adopt a time and cost effective
methodology which can be referred as In-vitro technique. Five methods were used to select the best
one and later that method was standardized. This may be used for pathogenicity test, to check the
Pathogen variability/ aggressiveness of the pathogen and preliminary screening of varieties/
germplasm against the pathogen. These methods include pot and test tube experiments by
application of inoculum and seed in soil simultaneously, by dipping roots in spore suspension
before planting in soil, by foliage spray with spore suspension, seed coating with spore suspension
before planting in test tube with cotton swab method. Out of these five procedures the test tube with
cotton swab method has been found the most appropriate and effective for the study of B.
sorokiniana.

Introduction

     Bipolaris sorokiniana (Cochliobolus sativus) is a world wide economically important
leaf blight / foliar spot causing pathogen of the wheat. It also causes root rot and black
point on grains (Wiese, 1998), most frequently associated with poor germination and
abnormal seedlings of wheat (Ammara et al., 2001). The fungus is one of the constraints
for crops in warmer growing areas and causing significant yield losses (Aftabuddin et al.,
1991). In north eastern and north western plains of India the yield losses ranged from 27 -
56.6% during 1998-99, incited due to the leaf blight caused by B. sorokiniana (Satvinder
et al., 2002; Singh et al., 2002). Pathogen has also been reported to be the one of
principal fungus involved in the seedling blight and root rot of wheat in Pakistan (Bhatti
et al., 1986; Hafiz, 1976; Kishwar et al., 1992).
     Different methods are being employed to study the In vitro disease development by
different workers in the research world. These methods include pot experiments by using
wheat seedlings and with different ways of inoculation (Lamari & Bernier, 1989),
plantlets regeneration of embryonic callus through In-vitro method (Chang et al., 1997)
and test tube method by using inverted cone of filter paper (Kishwar et al., 1992). To
study the seed and soil borne pathogen different methods have been tried to check the
pathogenic nature of the pathogen, its variability and aggressiveness and preliminary
screening of cultivars/ germplasm before going for identification of source of resistance
under natural conditions. The purpose of the current study was to standardize a procedure
416                                                           SHAMIM IFTIKHAR ET AL.,

by using the B. sorokiniana as a model organism having a seed and soil borne nature. The
method can be used to see the response of the host to the pathogen, variation of the
pathogen and its interaction to the host and varietal behaviour against the pathogen.

Materials and Methods

    Five different methods were used for pathogenicity test to select the method which
may be standardized for pathogenicity, pathogen variability/ aggressiveness analysis and
screening of varieties against the seed and soil borne pathogen. During the current study,
(Bipolaris sorokiniana) the most frequently isolated pathogen of leaf spot was used. The
methodologies are:

Seed with inoculum disk method: Wheat seeds disinfected with 1% Clorox were sown
in the autoclaved soil media (1:1:1 soil, sand and peat moss) in the plastic pots. Three
seeds / pot were sown with a 5 mm disk of 7 days old fungal culture (approximately
containing 3.2 x 104 spores/ disk). Disk was placed adjacent to seeds. The pots were
incubated at 22 - 250C in growth room by placing on the racks randomly. The data was
recorded after 30 days by noting the spots on leaves on 0-5 scale (Anon., 1996) and
confirmed the Koch’s postulate.

Root Dip method: Wheat seeds were sown in autoclaved sand and watered daily. After
6-8 days of the sowing, the raised seedlings were removed from the sand very carefully
so that roots remain intact. These seedlings were dipped in the spore suspension of B.
sorokiniana (3.2 x 104 spores /ml) and were planted in pots, already filled with
autoclaved soil containing silt loam, sand and peat moss (1:1:1). The pots were placed in
growth room at 22 - 250C and the symptoms observed after four weeks on 0-5 scale on
foliage (Anon., 1996) and confirmed the Koch’s postulate.

Spore suspension spray method: Pure culture of B. sorokiniana was obtained from
single spore culture maintained on PDA. The mycelium of the full grown culture was
scraped with a bent metal rod. Then conidial suspension was prepared by flooding the
plates with sterilized distilled water and the spore concentration was determined with a
haemocytometer (Lamari & Bernier, 1989). A drop of Tween-20 was added per 100ml of
suspension before application. Five wheat seeds of Wafaq-2001 were sown in plastic pots
containing sterilized soil (as prepared in above methods) and placed in a growth room. At
seedling stage (at 2-3 leaf stage after planting for 17-18 days) plants were sprayed with a
spore suspension containing 3.2 x 104 spore/ ml. Approximately 15 ml of inoculum was
sprayed per plant with a sprayer. The plants were covered with plastic bag for attaining
high humidity for 30 hours and then plants were placed in growth room at 22 - 25oC and
the disease severity observed as above and confirmed the Koch’s postulate.

Seed Coating method: Wheat seeds were surface sterilized with 1% Clorox and was
rinsed thrice with sterilized distilled water. Few drops of Tween-20 were sprayed on
these seeds and then were put on the actively grown culture of B. sorokiniana. The seeds
were mixed in the mature culture with the help of rod so that maximum spores may stick
with the seeds. Later these seeds were sown in the pots as in the above methods and
placed in growth chamber. Disease severity was observed on 0-5 scale upon the
appearance of spots on the leaves and confirmed the Koch’s postulate.
IN VITRO SCREENING OF WHEAT AGAINST BIPOLARIS SOROKINIANA                                 417

Test Tube Cotton Swab Method: Test tubes (20 cm x 3 cm) were prepared by filling 1/ 4th
of cotton in the bottom of the tube. Sterilized distilled water (20 ml) was added in each tube
and lids were covered with aluminum foil and were autoclaved. Wheat seeds (three
seeds/tube) were surface disinfected with 1% Clorox and placed on the moist cotton swab
in the test tube. One disk of 5 mm of fungal isolate (3.2 x 104 spores/ disk) was placed
adjacent to the seeds (Giri et al., 2001). After inoculation tubes were again covered with
aluminum foil and were placed in growth room at 25oC.

Standardization of method: Method was standardized by the application of two
treatments.

i. Inoculum with seeds: Described as above (Giri et al., 2001).
ii. Inoculum after initiation of roots: The seeds were first allowed to germinate on
cotton swab. When the roots appeared and seedlings became 5 days old, the inoculum
disc was placed on the roots. After 30 days when symptoms appeared, the data was
recorded in same fashion as above. The data was recorded on a revised (0-5) scale
(Anon., 1996). The revised scale was 0 = no symptoms, 1 = 1-5% spots on leaves, 2 = 6-
20% spots on leaves, 3 = 21-40% spots on leaves, 4 = 41-60% spots on leaves, 5 = 61%
and above spots on leaves. Later the data was subjected to analysis of variance and means
interaction by Computer Soft Ware Package M-Stat-C.

Results and Discussion

     Among the five In-vitro methods, four were conducted in pots and one in test tubes.
Out of five methods cotton swab test tube method (Fig. 1) was found the best one for
pathogenicity test and subsequently determined the most efficient method to be used for
pathogenicity/screening/aggressiveness analysis. The results revealed that there was no
significant effect of replications. However, there was a highly significant effect of isolate,
method and isolate x method interaction (Table 1). The mean values of different methods
revealed that M1 and M5 showed highly significant results as compared to one another
and the rest of the methods. However there was no significant difference between M2,
M3 and M4 (Table 2). The interactive means of the methods M1, M3 and M5 showed
highly significant difference from one another while M2 and M3 had slightly significant
difference. However M1 and M4 had no significant difference. The results showed that
M5 had highly significant difference compared to other methods (Table 3).
     After statistical analysis of the data it was found that the test tube moist cotton swab
method proved to be the most effective and efficient method for pathogenicity test and to
check the aggressiveness of the pathogen. The method is also more convenient for
preliminary screening of the varieties/ germplasm before going to field. In our studies this
method was specially compared with the method used by Lamari & Bernier (1989) which
was later adopted by Iram & Ahmad (2005), in which the plants at seedling stage planted
in pots needed to be sprayed with spore suspension with a particular concentration and
for the maintenance of humidity the pots with plants need to be covered with plastic bags
for 30 hours, whereas the test tube method proved to be more efficient as in this method
there is no need to maintain humidity. The method was taking the long time to develop
the symptoms and the symptoms developed were not as good as compared to the
symptoms developed in test tube method (Fig. 2). Another advantage of this method was
that the infection on the roots (if required) can be measured in the same experiment. So
the seed and soil borne pathogens can be easily tested by this method.
418                                                              SHAMIM IFTIKHAR ET AL.,




                           Fig. 1. Cotton Swab Test Tube Method




  Fig. 2. Symptom on wheat leaves during pathogenicity test by cotton swab test tube method.
IN VITRO SCREENING OF WHEAT AGAINST BIPOLARIS SOROKINIANA                                                          419

   Table 1. Analysis of variance of different methods applied for testing the most
             efficient one for pathogenicity test of Bipolaris sorokiniana.
 Sources of             Degree of        Sum, of                 Mean square
 variation               freedom         squares             Leaf             Root
 Replication                 4             1.00           0.250 NS          0.200 NS
 Isolates                    1             5.780           5.780**           3.920**
 Methods                     4            65.400          16.350**          13.400**
 Isolate x method            4             2.520           0.630**           0.720**
 Error                      36             7.800            0.217             0.078
 **Mean squares of isolates, methods and isolate x methods interaction are highly significant at probability level 1.

       Table 2. Mean value of different methods applied for pathogenicity of
                              Bipolaris sorokiniana.
                                                     Mean values
 S. No.         Methods
                                            Leaf                    Root
   1.             M1                       1.900B                  2.200B
   2.             M2                       1.300C                  2.400B
   3.             M3                       0.900C                   0.00C
   4.             M4                       2.200C                  2.4..B
   5.             M5                       4.200a                   3.00b
                LSD 5%                     0.4225                  0.2533
 Column means followed by a common letter are rot statistically different at 5% by LSD test
 *M1 = Seed + inoculum disk in the soil together.
 *M2 = Root dip of seedlings in spore suspension.
 *M3 = Spray of spores suspension on foliage at seedling stage.
 *M4 = Seed + conidia/ spores (coated on seed).
 *M5 = Test tube moist cotton swab method.

   Table 3. Mean value of interaction of different methods applied for testing the
                                most efficient one.
   S. No.        Methods              Leaf (0-5 scale)         Root (0-3 scale)
     1.             M1                     2.600c                    2.600
     2.             M2                    1.400de                  2.800ab
     3.             M3                     1.000e                    0.000
     4.             M4                     2.600a                   3.000a
     5.             M5                     4.600a                   3.000a
                LSD value                  0.5975                   0.3580
 Column means followed by a common letter are not statistically different at 5 %

References

Aftabuddin, A., W.E. Grey, D.E. Mathre and A.L. Scharen. 1991. Resistance in spring wheat to
     common root rot, spot blotch and black point caused by Cochliobolus sativus. Proc. First Int.
     Workshop on Common Root Rot of Cereals. Saskatoon, Canada. 11-14 August 1991. pp. 45-47.
Ammara, I., S.A. Anwar, R. Abid and Khan. M.S.A. 2001. Seed-borne pathogens associated with
     wheat seed and their role in poor germination. Pakistan Journal of Phytopathology, 13(2):
     102-106.
Anonymous. 1996. Standard evaluation system for rice. 4th Edition. International Rice Research
     Institute, Philippine.
420                                                                  SHAMIM IFTIKHAR ET AL.,

Bhatti, M.A.R. and M.B. Ilyas. 1986. Wheat diseases in Pakistan. In: Problems and progress of
     Wheat in South Asia, pp 20-30. (Eds.): L.M. Joshi, D.V. Singh and K.D. Srivastava, New
     Dehli, India. Malhotra publishing house. 401 pp.
Chang, Naitao, G. Zenggui, J. Xianlu, W. Wei and W. Yousan. 1997. In vitro selection for spot
     blotch resistance in wheat. In: Helminthosporium blights of wheat: Spot blotch and Tan spot.
     (Eds.): E. Duveiller, H.J. Dubin, J. Reeves and A. McNab Mexico, DF, Mexico: Centro
     Internacional de Mejoramiento de Maiz y Trigo (CIMMYT), pp. 253-258.
Giri, G.K., R.M. Gade and C.U. Petail. 2001.Seed borne Bipolaris sorokiniana in wheat and its
     chemical control. Journal of Soils and Crops, 11(1): 109-112.
Hafiz, A. 1986. Plant Diseases: Pakistan Agricultural Research Council. 552 pp.
Iram, S. and Ahmad, I. 2005. Prevalence and disease incidence of foliar blight of wheat in rice-
     wheat cropping system of Punjab. Pakistan Journal of Botany. 37(4): 973 – 980.
Kishwar, A., H. Sher, S. Iftikhar, K. Ali and S. Hassan. 1992. Foot rot diseases of wheat in rainfed
     areas of North West Frontier Provinces and Punjab. Sarhad Journal of Agriculture, 8: 541-
     545.
Lamari, L. and C.C. Bernier. 1989. Toxin of Pyrenophora tritici-repentis: host specificity,
     significance in disease and inheritance of host reaction. Phytopathology, 79(7): 740-744.
Ledingham, R.J., T.G. Atkinson, J. S. Horricks, J. T. Mills, L.J. Piening and R.D. Tinlane. 1973.
     Wheat losses due to common root rot in the Prairie Provinces of Canada, 1969-71. Canadian
     Plant Diseases Survey, 53: 113-122.
Satvinder, K., G.S. Nanda, G.M. Navneet, S. Kaur and N. Ghuman. 2002. Status of seed health and
     facultative foliar pathogens of wheat in Punjab from 1992-99. Journal of Research, Punjab
     Agricultural University. 2002, 39: 1, 28-34.
Singh, D.P., A.K. Sharma, S. Amerika, R.V. Singh, A.N. Tewari, A.K. Singh, R.N. Singh and S.P.
     Singh. 2002. Losses caused due to leaf blight in wheat in different agro climatic zones of
     India. Plant Diseases Research, 17(2): 313-317.
Wiese, M.V. 1998. Compendium of wheat diseases (3rd Ed). St. Paul, USA: APS Press, 112pp.

                             (Received for publication 15 June 2007)

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Jurnal koch

  • 1. Pak. J. Bot., 40(1): 415-420, 2008. SELECTION OF IN VITRO TECHNIQUE FOR PATHOGENICITY AND SCREENING OF WHEAT CULTIVARS AGAINST BIPOLARIS SOROKINIANA SHAMIM IFTIKHAR, SHAHZAD ASAD, ANJUM MUNIR AND IFTIKHAR AHMAD* Crop Diseases Research Program, Institute of Plant and Environmental Protection, National Agricultural Research Centre, Park Road, Islamabad-45500, Pakistan *Member Plant Sciences, PARC, P.O.Box, 1031, Islamabad Abstract Bipolaris sorokiniana (Cochliobolus sativus) is a seed and soil borne pathogen causing leaf blight or spot blotch of wheat worldwide. In Pakistan the pathogen was identified as predominant leaf spot causing fungus in wheat growing areas during 2004 – 2006. A lab based technique has been standardized to study the pathogenic nature of the pathogen, its variability/ aggressiveness and preliminary screening of cultivars/ germplasm before going to explore the sources of resistance under natural conditions. The purpose of this study was to adopt a time and cost effective methodology which can be referred as In-vitro technique. Five methods were used to select the best one and later that method was standardized. This may be used for pathogenicity test, to check the Pathogen variability/ aggressiveness of the pathogen and preliminary screening of varieties/ germplasm against the pathogen. These methods include pot and test tube experiments by application of inoculum and seed in soil simultaneously, by dipping roots in spore suspension before planting in soil, by foliage spray with spore suspension, seed coating with spore suspension before planting in test tube with cotton swab method. Out of these five procedures the test tube with cotton swab method has been found the most appropriate and effective for the study of B. sorokiniana. Introduction Bipolaris sorokiniana (Cochliobolus sativus) is a world wide economically important leaf blight / foliar spot causing pathogen of the wheat. It also causes root rot and black point on grains (Wiese, 1998), most frequently associated with poor germination and abnormal seedlings of wheat (Ammara et al., 2001). The fungus is one of the constraints for crops in warmer growing areas and causing significant yield losses (Aftabuddin et al., 1991). In north eastern and north western plains of India the yield losses ranged from 27 - 56.6% during 1998-99, incited due to the leaf blight caused by B. sorokiniana (Satvinder et al., 2002; Singh et al., 2002). Pathogen has also been reported to be the one of principal fungus involved in the seedling blight and root rot of wheat in Pakistan (Bhatti et al., 1986; Hafiz, 1976; Kishwar et al., 1992). Different methods are being employed to study the In vitro disease development by different workers in the research world. These methods include pot experiments by using wheat seedlings and with different ways of inoculation (Lamari & Bernier, 1989), plantlets regeneration of embryonic callus through In-vitro method (Chang et al., 1997) and test tube method by using inverted cone of filter paper (Kishwar et al., 1992). To study the seed and soil borne pathogen different methods have been tried to check the pathogenic nature of the pathogen, its variability and aggressiveness and preliminary screening of cultivars/ germplasm before going for identification of source of resistance under natural conditions. The purpose of the current study was to standardize a procedure
  • 2. 416 SHAMIM IFTIKHAR ET AL., by using the B. sorokiniana as a model organism having a seed and soil borne nature. The method can be used to see the response of the host to the pathogen, variation of the pathogen and its interaction to the host and varietal behaviour against the pathogen. Materials and Methods Five different methods were used for pathogenicity test to select the method which may be standardized for pathogenicity, pathogen variability/ aggressiveness analysis and screening of varieties against the seed and soil borne pathogen. During the current study, (Bipolaris sorokiniana) the most frequently isolated pathogen of leaf spot was used. The methodologies are: Seed with inoculum disk method: Wheat seeds disinfected with 1% Clorox were sown in the autoclaved soil media (1:1:1 soil, sand and peat moss) in the plastic pots. Three seeds / pot were sown with a 5 mm disk of 7 days old fungal culture (approximately containing 3.2 x 104 spores/ disk). Disk was placed adjacent to seeds. The pots were incubated at 22 - 250C in growth room by placing on the racks randomly. The data was recorded after 30 days by noting the spots on leaves on 0-5 scale (Anon., 1996) and confirmed the Koch’s postulate. Root Dip method: Wheat seeds were sown in autoclaved sand and watered daily. After 6-8 days of the sowing, the raised seedlings were removed from the sand very carefully so that roots remain intact. These seedlings were dipped in the spore suspension of B. sorokiniana (3.2 x 104 spores /ml) and were planted in pots, already filled with autoclaved soil containing silt loam, sand and peat moss (1:1:1). The pots were placed in growth room at 22 - 250C and the symptoms observed after four weeks on 0-5 scale on foliage (Anon., 1996) and confirmed the Koch’s postulate. Spore suspension spray method: Pure culture of B. sorokiniana was obtained from single spore culture maintained on PDA. The mycelium of the full grown culture was scraped with a bent metal rod. Then conidial suspension was prepared by flooding the plates with sterilized distilled water and the spore concentration was determined with a haemocytometer (Lamari & Bernier, 1989). A drop of Tween-20 was added per 100ml of suspension before application. Five wheat seeds of Wafaq-2001 were sown in plastic pots containing sterilized soil (as prepared in above methods) and placed in a growth room. At seedling stage (at 2-3 leaf stage after planting for 17-18 days) plants were sprayed with a spore suspension containing 3.2 x 104 spore/ ml. Approximately 15 ml of inoculum was sprayed per plant with a sprayer. The plants were covered with plastic bag for attaining high humidity for 30 hours and then plants were placed in growth room at 22 - 25oC and the disease severity observed as above and confirmed the Koch’s postulate. Seed Coating method: Wheat seeds were surface sterilized with 1% Clorox and was rinsed thrice with sterilized distilled water. Few drops of Tween-20 were sprayed on these seeds and then were put on the actively grown culture of B. sorokiniana. The seeds were mixed in the mature culture with the help of rod so that maximum spores may stick with the seeds. Later these seeds were sown in the pots as in the above methods and placed in growth chamber. Disease severity was observed on 0-5 scale upon the appearance of spots on the leaves and confirmed the Koch’s postulate.
  • 3. IN VITRO SCREENING OF WHEAT AGAINST BIPOLARIS SOROKINIANA 417 Test Tube Cotton Swab Method: Test tubes (20 cm x 3 cm) were prepared by filling 1/ 4th of cotton in the bottom of the tube. Sterilized distilled water (20 ml) was added in each tube and lids were covered with aluminum foil and were autoclaved. Wheat seeds (three seeds/tube) were surface disinfected with 1% Clorox and placed on the moist cotton swab in the test tube. One disk of 5 mm of fungal isolate (3.2 x 104 spores/ disk) was placed adjacent to the seeds (Giri et al., 2001). After inoculation tubes were again covered with aluminum foil and were placed in growth room at 25oC. Standardization of method: Method was standardized by the application of two treatments. i. Inoculum with seeds: Described as above (Giri et al., 2001). ii. Inoculum after initiation of roots: The seeds were first allowed to germinate on cotton swab. When the roots appeared and seedlings became 5 days old, the inoculum disc was placed on the roots. After 30 days when symptoms appeared, the data was recorded in same fashion as above. The data was recorded on a revised (0-5) scale (Anon., 1996). The revised scale was 0 = no symptoms, 1 = 1-5% spots on leaves, 2 = 6- 20% spots on leaves, 3 = 21-40% spots on leaves, 4 = 41-60% spots on leaves, 5 = 61% and above spots on leaves. Later the data was subjected to analysis of variance and means interaction by Computer Soft Ware Package M-Stat-C. Results and Discussion Among the five In-vitro methods, four were conducted in pots and one in test tubes. Out of five methods cotton swab test tube method (Fig. 1) was found the best one for pathogenicity test and subsequently determined the most efficient method to be used for pathogenicity/screening/aggressiveness analysis. The results revealed that there was no significant effect of replications. However, there was a highly significant effect of isolate, method and isolate x method interaction (Table 1). The mean values of different methods revealed that M1 and M5 showed highly significant results as compared to one another and the rest of the methods. However there was no significant difference between M2, M3 and M4 (Table 2). The interactive means of the methods M1, M3 and M5 showed highly significant difference from one another while M2 and M3 had slightly significant difference. However M1 and M4 had no significant difference. The results showed that M5 had highly significant difference compared to other methods (Table 3). After statistical analysis of the data it was found that the test tube moist cotton swab method proved to be the most effective and efficient method for pathogenicity test and to check the aggressiveness of the pathogen. The method is also more convenient for preliminary screening of the varieties/ germplasm before going to field. In our studies this method was specially compared with the method used by Lamari & Bernier (1989) which was later adopted by Iram & Ahmad (2005), in which the plants at seedling stage planted in pots needed to be sprayed with spore suspension with a particular concentration and for the maintenance of humidity the pots with plants need to be covered with plastic bags for 30 hours, whereas the test tube method proved to be more efficient as in this method there is no need to maintain humidity. The method was taking the long time to develop the symptoms and the symptoms developed were not as good as compared to the symptoms developed in test tube method (Fig. 2). Another advantage of this method was that the infection on the roots (if required) can be measured in the same experiment. So the seed and soil borne pathogens can be easily tested by this method.
  • 4. 418 SHAMIM IFTIKHAR ET AL., Fig. 1. Cotton Swab Test Tube Method Fig. 2. Symptom on wheat leaves during pathogenicity test by cotton swab test tube method.
  • 5. IN VITRO SCREENING OF WHEAT AGAINST BIPOLARIS SOROKINIANA 419 Table 1. Analysis of variance of different methods applied for testing the most efficient one for pathogenicity test of Bipolaris sorokiniana. Sources of Degree of Sum, of Mean square variation freedom squares Leaf Root Replication 4 1.00 0.250 NS 0.200 NS Isolates 1 5.780 5.780** 3.920** Methods 4 65.400 16.350** 13.400** Isolate x method 4 2.520 0.630** 0.720** Error 36 7.800 0.217 0.078 **Mean squares of isolates, methods and isolate x methods interaction are highly significant at probability level 1. Table 2. Mean value of different methods applied for pathogenicity of Bipolaris sorokiniana. Mean values S. No. Methods Leaf Root 1. M1 1.900B 2.200B 2. M2 1.300C 2.400B 3. M3 0.900C 0.00C 4. M4 2.200C 2.4..B 5. M5 4.200a 3.00b LSD 5% 0.4225 0.2533 Column means followed by a common letter are rot statistically different at 5% by LSD test *M1 = Seed + inoculum disk in the soil together. *M2 = Root dip of seedlings in spore suspension. *M3 = Spray of spores suspension on foliage at seedling stage. *M4 = Seed + conidia/ spores (coated on seed). *M5 = Test tube moist cotton swab method. Table 3. Mean value of interaction of different methods applied for testing the most efficient one. S. No. Methods Leaf (0-5 scale) Root (0-3 scale) 1. M1 2.600c 2.600 2. M2 1.400de 2.800ab 3. M3 1.000e 0.000 4. M4 2.600a 3.000a 5. M5 4.600a 3.000a LSD value 0.5975 0.3580 Column means followed by a common letter are not statistically different at 5 % References Aftabuddin, A., W.E. Grey, D.E. Mathre and A.L. Scharen. 1991. Resistance in spring wheat to common root rot, spot blotch and black point caused by Cochliobolus sativus. Proc. First Int. Workshop on Common Root Rot of Cereals. Saskatoon, Canada. 11-14 August 1991. pp. 45-47. Ammara, I., S.A. Anwar, R. Abid and Khan. M.S.A. 2001. Seed-borne pathogens associated with wheat seed and their role in poor germination. Pakistan Journal of Phytopathology, 13(2): 102-106. Anonymous. 1996. Standard evaluation system for rice. 4th Edition. International Rice Research Institute, Philippine.
  • 6. 420 SHAMIM IFTIKHAR ET AL., Bhatti, M.A.R. and M.B. Ilyas. 1986. Wheat diseases in Pakistan. In: Problems and progress of Wheat in South Asia, pp 20-30. (Eds.): L.M. Joshi, D.V. Singh and K.D. Srivastava, New Dehli, India. Malhotra publishing house. 401 pp. Chang, Naitao, G. Zenggui, J. Xianlu, W. Wei and W. Yousan. 1997. In vitro selection for spot blotch resistance in wheat. In: Helminthosporium blights of wheat: Spot blotch and Tan spot. (Eds.): E. Duveiller, H.J. Dubin, J. Reeves and A. McNab Mexico, DF, Mexico: Centro Internacional de Mejoramiento de Maiz y Trigo (CIMMYT), pp. 253-258. Giri, G.K., R.M. Gade and C.U. Petail. 2001.Seed borne Bipolaris sorokiniana in wheat and its chemical control. Journal of Soils and Crops, 11(1): 109-112. Hafiz, A. 1986. Plant Diseases: Pakistan Agricultural Research Council. 552 pp. Iram, S. and Ahmad, I. 2005. Prevalence and disease incidence of foliar blight of wheat in rice- wheat cropping system of Punjab. Pakistan Journal of Botany. 37(4): 973 – 980. Kishwar, A., H. Sher, S. Iftikhar, K. Ali and S. Hassan. 1992. Foot rot diseases of wheat in rainfed areas of North West Frontier Provinces and Punjab. Sarhad Journal of Agriculture, 8: 541- 545. Lamari, L. and C.C. Bernier. 1989. Toxin of Pyrenophora tritici-repentis: host specificity, significance in disease and inheritance of host reaction. Phytopathology, 79(7): 740-744. Ledingham, R.J., T.G. Atkinson, J. S. Horricks, J. T. Mills, L.J. Piening and R.D. Tinlane. 1973. Wheat losses due to common root rot in the Prairie Provinces of Canada, 1969-71. Canadian Plant Diseases Survey, 53: 113-122. Satvinder, K., G.S. Nanda, G.M. Navneet, S. Kaur and N. Ghuman. 2002. Status of seed health and facultative foliar pathogens of wheat in Punjab from 1992-99. Journal of Research, Punjab Agricultural University. 2002, 39: 1, 28-34. Singh, D.P., A.K. Sharma, S. Amerika, R.V. Singh, A.N. Tewari, A.K. Singh, R.N. Singh and S.P. Singh. 2002. Losses caused due to leaf blight in wheat in different agro climatic zones of India. Plant Diseases Research, 17(2): 313-317. Wiese, M.V. 1998. Compendium of wheat diseases (3rd Ed). St. Paul, USA: APS Press, 112pp. (Received for publication 15 June 2007)