Investigation was conducted to examine the pathogenic variability of the isolates of Bipolaris sorokiniana at three growth stages viz. seedling stage, tillering stage and heading stage of two wheat varieties (BARI Gom 26 and Kanchan). Based on the morphological variation of seven isolates of B. sorokiniana, BdBSLtan 6 was the most potent considering the maximum mycelial growth (5.40 cm), and the highest conidia production capacity (13.3×106 conidia/mL). At each growth stage, three selected isolates (BdBSL_tan, BdBSL_mym and BdBSL_jam) were inoculated and BdBSL_tan 6 showed the most potential to cause higher infection (100%) in both varieties followed by BdBSL_mym 2. Tillering stage was most vulnerable showing the highest incidence and severity which was followed by heading stage. It canconclusively suggest that both the Bangladeshi isolates (BdBSL_tan and BdBSL_mym) are morphologically distinct and can be aggressive to cause infection in wheat plant at tillering stage and heading stage.
Wheat (Triticum aestivum L.) occupies a position of paramount importance amongst world’s crop plants, providing nearly 20% of the total world food requirement. It is considered as the second most important staple food crop next to rice in Bangladesh. The fungal pathogens are of most significance and take a regular toll of the yield wherever wheat is sown in the country. There are some pathogens which are highly seed-borne and seed-transmitted in wheat [1]. Among all the devastating pathogens of wheat Bipolaris sorokiniana reported to be the most significant in disease development lowering wheat yield in the country [2]. The pathogen has also been reported to be highly seed-borne as well as soil-borne and air- borne. Bipolaris sorokiniana plays a vital role for causing leaf blight, leaf spot and black point of wheat disease [3,4]. The disease is characterized by brown to black discoloration usually restricted to the embryonic end of the grain, but in case of severe infection, the whole grain may be discolored and shriveled [5]. In Bangladesh, the prevalence of the disease was found to vary from 5-55% depending on different varieties grown in the major wheat growing areas [2]. Black point has an adverse effect on seed weight, germination, and seedling emergence [6,7]. However, the reduction in grain weight and germination is found to be related with the severity of black point infection. Increased seedling mortality and reduced seedling vigor and grain yield are also experienced when black pointed seeds are used for sowing [1,8,9]. The germination of black point affected seed was reduced to a great extent compared to the healthy grains [6]. Moreover, the reduction of germination was found in respect to the severity of black point infection [10,11].
Since the black point disease of wheat causes significant yield loss, reduces the quality of the seed and has appeared as a potential threat for seed producer as well as to the farmers in Bangladesh. Black point also affects luster and plumpness of wheat grain and reduces its market value [12]. The disease starts at the seedling stage and increases with the plant age [2]. Leaf blight / spot and black point caused by B. sorokiniana has become a serious concern in Bangladesh [13]. In case of severe attack, it may result in 100% yield loss [8]. Leaf blight disease is considered to be a major threat to the wheat cultivation all over the world [14]. Leaf blight severity increased in the field with the increasing level of seed infection in different established wheat varieties in the country [1].
The above discussion indicates the possible presence of variation in the population of B. sorokiniana in terms of virulence and aggressiveness. Previous study also showed the presence of several strains and races having different degree of pathogenicity in Bangladesh [15,16]. Therefore, different races can cause different levels of disease which is very important to know to take necessary measurement for controlling the disease. Very little or no work has been done to evaluate the pathogenic variability in causing differential disease severity in wheat by B. sorokiniana. The present work was therefore, undertaken to observe the variation of different isolates of B. sorokiniana collected from major wheat growing areas of Bangladesh and identify the most vulnerable growth stage of wheat to B. sorokiniana.
Experimental Section
Wheat seed samples were collected from major wheat growing areas of Bangladesh e.g. Mymensingh, Gazipur, Dinajpur, Kurigram, Pabna, Tangail and Jamalpur. To isolate B. sorokiniana from seed, the seeds first incubated on moist blotter paper following the method of International Seed Testing Association [17]. In this method, the seeds were placed on three layered moistened blotter paper in petridishes. The petridishes were incubated at room temperature under Near Ultra Violate (NUV) light for 7 days. Then the B. sorokiniana associated with the seeds were identified and recorded by observing the growth characters on the incubated seeds under stereo-binocular microscope following the key outlined by Chidaimbaram. Single conidium of B. sorikiniana from the incubated seeds was transferred under stereo-binocular microscope onto potato dextrose agar (PDA) medium with the help of an inoculation loop and incubated at 25±2ºC for about a week with 12h photoperiod. Successive subcultures were made for getting pure cultures of the isolates. The isolates were studied for the morphological characteristics. Inoculum from each of the isolates was transferred to slant (test tube containing PDA) and incubated as before and stored at 4°C for future study. For morphological study data were collected on radial mycelial growth, colony color, texture, conidial size, shape and conidial concentration by using haemacytometer.
To evaluate the pathogenic variability of the isolates pot experiments were conducted using earthen pot containing soil collecting from nearby locations of non-wheat cultivated agricultural land of Mymensingh. Wheat Seeds of Kanchon and BARI Gom 26 were collected from Wheat Research Centre, Dinajpur. The earthen pots of 30 cm diameter were filled with soil mixed thoroughly with compost (4:1), TSP and MoP fertilizer. Wheat plants at three growth stages viz. seedling, tillering and heading stage were subjected for artificial inoculation with the B. sorokiniana suspension using a hand sprayer. Based on the morphology, conidial density and spore potency three representative isolates viz. BdBSL_mym, BdBSL_tan and BdBSL_jam were considered for inoculating the wheat plants for having pathogenic response of the isolates. A conidial suspension having 104conidia/ml was applied for inoculation according to the method developed by CIMMYT [18]. The wheat plants were covered with previously moistened polythene bags for 36 hours for maintaining moisture immediately after inoculation. After removing the polythene bags the leaf surface were exposed but watering of pots was continued for supplying moisture to the seedlings [19]. Ten plants were selected at random and tagged in each pot on the onset of disease development. Disease index rated by 0-5 scale outlined by Bazlur Rashid, [20] was followed.
Disease incidence and severity were calculated by using these formulae:
Statistical Analysis
Both laboratory and net houseexperiments were done by using Completely Randomized Design (CRD). All treatments were replicated thrice. The collected data were analyzed by using MSTAT C statistical software and the mean differences were evaluated at P = 0.05 and P = 0.01 levels of significance by Duncun’s Multiple Range Test (DMRT).
Table 1: Ratings for Leaf Blight Severity by B. Sorokinianaon Wheat [20]
| Ratings/Grade | Lesion | ||
Type with foliar damage | Size (mm) | Number Per Leaf | |
0 | No lesion | - | - |
1 | Light to dark brown usually with yellowish halo and restricted to less than 1% of the leaf blade | 1 – 2 | 1 – 2 |
2 | Light to dark brown usually with yellowish halo and covering up to not more than 5% of the leaf blade | 3 – 4 | 3 – 5 |
3 | Light brown with yellowish halo coalesced together and covering not more than 20% of the leaf blade | 5- 10 | 6 – 15 |
4 | Light brown to brown with yellow halo and covering not more than 75% of the leaf blade and 25% of the leaf sheath | >10 | >15 |
5 | Brown and covering more than 75% of the leaf sheath.Often the whole plant including panicle become blighted | >10 | >15 |
Morphological variation of the isolates: Different morphological traits of the isolates of B. sorokiniana were observed in in vitro study (Table 2). Diverse morphological phenotypes considering colony color, texture, conidial size, sporulation potency and radial mycelial growthamong the isolates were found. The isolates of B. sorokinian were isolated from infected wheat seeds collected from seven major wheat growing areas of Bangladesh viz. Gazipur, Mymensingh, Dinajpur, Kurigram, Pabna, Tangail and Jamalpur. Color of the pure culture of the isolates differentiated into black, whitish and ash.The upper surfaces of the pure culture were differed in texture ranging loose, compact and moderately compact. Mean radial mycelial growth of the isolates recorded at 9th day varied significantly. The most rapid mycelial growth (5.40 cm) was found in BdBSL_tan while the slow mycelial growth (2.90) was recorded in BdBSL_kur. Significant variation was also found in conidial density and ranged from 1×106 to 13.30×106/mL. The highest conidial density was recorded in BdBSL_tan and the lowest was in BdBSL_pab. The sporulation potency (conidia/cm2) of seven isolates differed significantly and ranged from 7.05×106 to 27.13×106. The maximum sporulation potency (conidia/cm2) was recorded in the isolate BdBSL_tan and the lowest sporulation potency (conidia/cm2) was recorded in the isolate BdBSL_gaz. Variation was also found in size and shape of the conidia.The largest conidia were observed in BdBSL_mym (6.0 μ X 1.19 μ) and the tiniest conidia were found in BdBSL-dij and BdBSL_kur (3.99 μ X 0.95 μ). Table 2 shows the variation in morphological characters of the isolates of bipolarissorokiniana.
Table 2: Variation in Morphological Characters of the Isolates of Bipolarissorokiniana
| SL No. | Isolates | Location | Colour | Texture | Radial Mycelial Growth (cm) | Density (conidia/ml) ×106 | Length ( m) | Breadth(
|
1 | BdBSL_gaz 1 | Gazipur | Ash | Loose | 3.65 c | 1.25 e | 5.0 | 1.0 |
2 | BdBSL_mym 2 | Mymensingh | Black | Compact | 3.00 d | 2.50 d | 6.0 | 1.19 |
3 | BdBSL_dij 3 | Dinajpur | Ash | Compact | 3.85 c | 2.75 d | 3.88 | 0.95 |
4 | BdBSL_kur 4 | Kurigram | Whitish | Loose | 2.90 d | 4.00 c | 3.88 | 0.95 |
5 | BdBSL_pab 5 | Pabna | Black | Compact | 3.10 d | 1.00e | 4.0 | 1.12 |
6 | BdBSL_tan 6 | Tangail | Ash | Loose | 5.40 a | 13.30a | 4.5 | 1.11 |
7 | BdBSL_jam 7 | Jamalpur | Black | Moderately compact | 5.05 b | 11.50b | 4.15 | 1.14 |
Responses of wheat seedlings cv. BARI Gom 26 to the isolates of B. sorokiniana at different growth stages: Effect of different isolates of B. sorokinianaon development of leaf blight/spot disease on wheat cultivar BARI Gom 26 inoculated at three different growth stages showed significant differences (Table 3). The percentage of disease incidence varied significantly at every growth stage compared to untreated control. The plants inoculated at seedling stage showed the minimum incidence for all the isolates, while the incidence was maximum at tillering stage followed heading stage. BdBSL_tan showed the highest virulence at every growth stage causing the highest incidence. Hundred percent plants were infected by BdBSL_tan at tillering and heading stage showing its highest virulence. The isolate BdBSL_mym also showed significantly higher incidences both at tillering stage (98.0%) and heading stage (86.67%). The un-inoculated plants showed higher incidences (30.0 %) at tillering stage indicating the seed-borne nature of the pathogen as well as the most vulnerable stage of wheat plants.
The severity of leaf blight/spot in BARI Gom26 due to inoculation by different isolates of B. sorokiniana varied significantly in different growth stages. Among the three stages; tillering stage was most vulnerable showing the highest severity for all the isolates. BdBSL_tan showed highest severity at all growth stages expressing the highest (86.33 %) at tillering stage followed by seedling stage (58.00 %). Without inoculation the plants showed higher (20.00 %) severity at tillering stage indicating some infection may be due to seed-transmitted nature of the pathogen. Table 3 shows incidence and severity of leaf blight/spot of wheat cv. bari gom 26 inoculated at different growth stages by different isolates of b. sorokiniana.
Table 3: Incidence and Severity of Leaf Blight/Spot of Wheat Cv. BARI Gom 26 Inoculated at Different Growth Stages by Different Isolates of B. Sorokiniana
| Treatment | Disease Incidence (%) | Disease Severity (%) | ||||
Seedling | Tillering | Heading | Seedling | Tillering | Heading | |
Control | 0.00 d | 30.00 b | 1.67 d | 0.00 d | 20.00 c | 0.67 d |
BdBSL_mym | 68.00 b | 98.00 a | 86.67 b | 44.00 b | 60.00 b | 43.00 b |
BdBSL_tan | 86.00 a | 100.0 a | 100.0 a | 58.00 a | 86.33 a | 56.00 a |
BdBSL_jam | 56.00 c | 95.00 a | 70.00 c | 35.00 c | 53.00 b | 30.00 c |
LSD0.05 | 5.61 | 4.89 | 11.65 | 3.25 | 10.01 | 3.44 |
Figures in a column with common letters do not differ significantly at 5% level of significance
Responses of wheat seedlings cv. Kanchan to the isolates of B. sorokiniana at different growth stages: Significant variations on development of leaf spot/blight by different isolates of B. sorokiniana were observed at three growth stages of wheat cv.Kanchan (Table 4). The isolates BdBSL_tan and BdBSL_mym showed higher aggressiveness causing infection at three growth stages. These two isolates cause 100% infection at tillering stage, while BdBSL_tan caused 100% infection both at tillering and heading stage. The un-inoculated plants showed 40% incidence indicating the seed-transmitted nature of B. sorokiniana. Table 4 shows the incidence and severity of leaf blight/spot of wheat cv. kanchan inoculated at different growth stages by different isolates of b. sorokiniana.
The severity of leaf spot/blight varied significantly at three stages of plant growth inoculated by different isolates of B. sorokiniana (Table 4). The severity of the disease is more extensive at tillering stage. The highest severity (86.33%) of the disease was found at tillering stage by BdBSL_mym followed by BdBSL_tan (70.00 %) at heading stage. The wheat plants without inoculation showed 20% disease severity indicating the seed-transmissibility of the pathogen which was reduced 4% at heading stage.
Table 4: Incidence and Severity of Leaf Blight/Spot of Wheat Cv. Kanchan Inoculated at Different Growth Stages by Different Isolates of B. Sorokiniana
| Treatment | Disease Incidence | Disease Severity | ||||
| Seedling | Tillering | Heading | Seedling | Tillering | Heading | |
| Control | 0.00 c | 40.00 b | 8.33 c | 0.00 d | 20.00 c | 4.00 d |
| BdBSL_mym | 86.00 a | 100.0 a | 96.67 a | 60.00 b | 86.33 a | 60.00 b |
| BdBSL_tan | 87.00 a | 100.0 a | 100.0 a | 67.00 a | 68.33 b | 70.00 a |
| BdBSL_jam | 71.00 b | 95.00 a | 91.67 b | 44.67 c | 66.33b | 43.33 c |
| LSD0.05 | 5.96 | 7.45 | 4.71 | 4.11 | 8.07 | 7.47 |
Figures in a column with common letters do not differ significantly at 5% level of significance
The pathogenic variability of B. sorokiniana at different growth stages of wheat was studied. Seven isolates of B. sorokiniana collected from different locations were studied on the basis of their morphological and physiological characteristics and inoculated onto two cultivars of wheat to investigate incidence and severity of leas spot/blight. The present study clearly indicate that variations exist among the isolates of B. sorokiniana of wheat seeds collected from different locations of Bangladesh considering morphological characters like redial mycelial growth, conidia size, conidia production capacity etc. The findings of the present study are in agreement with the study of Ahmed et al. [15], Monjil and Hossain [21] and Kohli and DiazdeAckermann, [22], where they reported the morphological variation among the isolates of B. sorokiniana. In the present study, in control (without inoculation) treatments, both susceptible check (Kanchan) and moderately resistant (BARI Gom 26) cultivar did not show any leaf spot/blight symptom at seedling stage. Conversely, under inoculated condition both varieties developed disease symptomat various level. These results indicate that if inoculum is available then disease severity can reach up to 67% in susceptible variety and 58% in moderately resistant variety BARI Gom 26 at seedling stage. The findings of the present study corroborate with the findings of Monjil and Hossain [21] where they reported that the highest leaf area diseased at one leaf stage after 48h, 72h, and 96 hours of inoculation were 40%, 75% and 95% respectively. Moreover, the highest leaf area disease at three leaf stages after 48h, 72h, and 96 hours of inoculation were 30%, 40% and 85% respectively. The findings of the present study are in agreement with the previous study of Ahmed et al. [15], Monjil and Hossain [21] and Kohli and Diazde Ackermann, [22], where they also reported the morphological variation among the isolates of B. sorokiniana.
At tillering stage, the plants in the control treatment showed disease symptoms and the incidence was 40% in cv. Kanchan whereas 100% incidence was recorded in both the varieties under inoculated condition. The results indicate that there is possibility of causing infection by seed transmission of B. sorokiniana which take some time to express symptoms. These results further indicate that tillering and heading stages are vulnerable for getting infection. It can be anticipated that suitable weather, susceptible variety and plant age are crucial for disease development in case of leaf spot/blight of wheat caused by B. sorokiniana. The present study is in agreement with Shaner [23] where it been reported that foliar wheatblotches are introduced into uninfected fields through infected seed. On the other hand, Ahmed et al. reported the variation among the pathotypes of B. sorokiniana causing leaf blight severity under inoculated condition ranging from 61 to 81 % in wheat cultivar Kanchan supports our present findings.
Significant incidence and severity of leaf spot/blight were recorded at heading stage for both the varieties indicating that like tillering stage heading stage is also very crucial for leaf blight development. Sing et al. [24] reported that foliar blight pathogen B. sorokiniana of wheat showing average incidence of about 62% at heading stage which is in conformity with the present findings. It is clear that among the three-inoculation stage, the tillering stage and heading stage are vulnerable for disease development. Disease development at different growth stages of wheat has a significant relationship with morpho-physiological characteristics of B. sorokiniana suggest that isolate with high growth, high conidial density and high sporulation potency causes more diseases compare to others. BdBSL-tan showed higher mycelial growth and conidia production and eventually yielded higher disease incidence and severity in susceptible check (Kanchan) or moderately resistant (BARI Gom 26) variety.
Isolates of B. sorokiniana of infected wheat seeds collected from seven wheat growing areas of Bangladesh have shown distinct morphological and physiological variation. Three morpho-physiologically distinct isolates were inoculated at three important growth stages of wheat to investigate their effect on incidence and severity in two wheat cultivars. The findings of the study indicate that the leaf spot/blight can be appeared at any growth stage, but tillering and heading stage in most vulnerable. All isolates of B. sorokiniana are pathogenic to both popular cultivars [16] of wheat, but strong variations are seen causing disease symptoms at different growth stages of plant growth. Based on these findings it is suggested that management strategies should be designed considering the host growth stage, seed-transmissibility and physiological traits of B. sorokiniana.
Acknowledgment
The study was funded by the Ministry of Science and Technology (MoST), The peoples’ Republic of Bangladesh.
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