Morphological and physiological variation among different isolates of Alternaria spp. from Rapeseed-Mustard

To find out the Morphological variation on growth and sporulation of Alternaria species of Alternaria leaf blight of mustard from 10 representative geographical locations of Bangladesh, this experiment was conducted at Plant Pathology Laboratory, Oilseed Research center, Bangladesh Agricultural Research Inistitute (BARI), Joydevpur, Gazipur, Bangladesh. All the isolates showed high level of variability in in-vitro in respect of radial mycial growth, colony colour, sub surface colour, colony shape, colony texture, zonation (surface and sub surface), length and width of conidia, beak length and number of septa. The maximum and minimum radial mycial growth was recorded 90 mm in isolate NATAb and 83.67 mm in isolate GAZAb, respectively at 14 days after incubation. Significant variation in conidial length, width, beak and no. of conidia observed in all isolates. The length of conidia ranged from 41.56 to 117.54µm with 3 to 11 transverse and 0 to 3 vertical septa. The width and beak length varied from 10.34 to 23.12 µm and 16.78 to 72.65 µm ,respectively. Surface colour were olivacious green to black and circular shaped colonies were observed in all isolates on PDA medium. Colony texture were cottony to velvety. Subsurface colour varied from light brown to black and pinkish. Zonation found in some isolates and some did not produce on both surface and subsurface. All conidia were murifrom and light brown to deep brown in colour. Potato Carrot Dextrose Agar medium (PCDA) and 25 o C temperature were found optimum for different isolates for mycelial growth and sporulation.


I. INTRODUCTION
Rapeseed-Mustard (Brassica spp.) is the principal oilproducing crop of Bangladesh yielding 77.51% [4] of total oilseed production from 60.3% of the total area coverage. This crop is cultivated, at present, in about 802882 acres. The production is about 359452 lac metric tons oil [4]. The average yield of mustard is 447 Kg/ha. Total production and per hectare seed yield of this crop may be increased by using high yielding variety (HYV) and improved production technologies. Rapeseed-mustard is cultivated almost all over the world. It is grown in tropical as well as temperate agro climatic zones and are the best adapted to areas having a relatively cool, moist climate during the growing season. [18]. Alternaria leaf blight caused by Alternaria brassicae is one of the major diseases of mustard [21,29,15,1,10,37,7]. This disease reduces mustard yield up to 47% [30] in India. It is a prominent disease in India, Australia, Canada, Africa, England, Germany, France, Sri Lanka, Spain and Sweden, all most all around the world [12]. Around the initial site of host leaf Alternaria morphologically produces a series of concentric rings [2]. A. brassicae is a necrotrophic pathogen produce lesion on leaves, stem and siliquae which affect seed quantity, quality by reducing oil content, seed size and seed colour [8]. This disease may cause significant losses in both temperate and tropical Brassica crops [20]. The major aspects of biology of an organism are the morphological and physiological characters of an individual within a species. Although, it is not frequent in asexually produced individual of the progeny. Variability studies are important to document the changes occurring in populations and individuals as variability in morphological and physiological traits indicate the existence of different pathotypes [21]. Anamorph form of this pathogen shows great variability in morphology, physiology and pathogenicity. Several researchers have reported existence of variability based on morphology, sporulation, growth and cultural characteristics. We know, every pathogen species has numerous biotypes, races or pathotype with specific genes in the respective host plants [36]. Proper understanding in the variation of pathogen population is highly crucial in the process of breeding for resistance against a particular disease. Considering the above fact this research was undertaken to Find out the morphological and physiological variation among different isolates of Alternaria spp.

Designation of collected isolates
The collected isolates were designed as DHAAb, GAZAb, MYMAb based on their collected location. For example an isolate collected from Dhaka and recognized as first three letters of the area and Ab indicate Alternaria brassicae (Table 1).

Preparation of Potato-Carrot Dextrose Agar (PCDA)
The combination of Potato-Carrot dextrose agar prepared by 100ml potato+100ml carrot extract, 1000ml distilled water, 17 gm agar, 20 gm dextrose in a conical flask and autoclaved at 121 c under 15 psi for 30 minutes. After autoclaved the media was kept few minutes for cool and added 25-30 drops of lactic acid then poured into sterile petriplates.

Isolation and Identification of Alternaria spp.
The pathogen was isolated by tissue planting method and incubated at 25±1˚ C for 7 days. After incubation the fungus mycelia were examined under stereomicroscope (Model: Motic, SMZ-168) & compound microscope (Model: Omano, OMTM-85) for identification of the pathogen. The fungus was identified following the keys of Eills [9] .

Purification and preservation of the pathogen
The pure culture of A. brassicae from the PDA was transferred to PDA slants and allowed to grow at 25+ 1 0 C for 7 days. After incubation PDA slants were preserved in refrigerator at 4 0 C for further study.
All the isolates were studied for morphological variations.
In terms of conidia color, shape, size, septation, was observed on PDA medium.

Effect of culture media on growth, spore production and time of sporulation
Mycelial discs of 7 days old culture of Alternaria spp. isolates were transferred to the center of PDA, CDA and PCDA and incubated at 25˚C and 22±1˚C and data were recorded on growth, spore production and time of sporulation. 3 replications were maintained for each isolates in a completely randomized design. The colony diameter was recorded on 2, 4, 6, 8, 10, 12, 14 days after inoculation.

Data Analysis
For cultural, morphological and the treatment means the data were statistically analyzed by Duncan's Multiple Range test (DMRT) with significance level at 5% [13]. The package used for analysis was MSTAT-C version -88, developed by Michigan State University, Agricultural University of Norway [11].

Colony characters of isolates of Alternaria spp. on PDA
Variation was observed in colony characters of 10 isolates of A. brassicae like surface colour, shape, texture, zonation and subsurface colour are presented Table 2 and Figure 1.

Morphological variation of conidia of different isolates of Alternaria spp. 3.2.1
Size of conidia of Alternaria spp. on PDA Remarkable variation was observed in length, breadth and beck size of conidia of different isolates of A. brassicae on PDA ( Table 3). The length of conidia of different isolates varied from 41.56µm to 117.54µm. The maximum mean length was recorded at MYMAb 113.1µm. The minimum length was recorded at isolates GAZAb 63.63µm. The breadth of conidia of different isolates varied from 10.34 µm to 23.12 µm. The maximum mean breadth was recorded at PABAb 17.36 µm. The minimum mean breadth was recorded at LALAb 20.29 µm.The beak of conidia of different isolates varied from 16.78 µm to 72.65 µm. The maximum mean beak was recorded at PABAb43.26 µm. The minimum mean beak was recorded at GAZAb 24.84 µm.  Variation in septation observed in isolates of A.brassicae. The horizontal septation varied from 3-7 to 7-11. The vertical septation varied from 0-1to 2-3. The maximum horizontal septation observed isolates BOGAb (7-11) and minimum septation observed in isolates GAZAb.The maximum vertical (2-3) septation observed in isolates MYMAb, RANAb and NATAb. The minimum vertical(0-1) septation observed in isolate DHAAb.

Radial mycelial growth of 10 isolates of Alternaria spp. on PCDA
After 2 days of inoculation the maximum radial mycelial growth of Alternaria spp. was measured in RAJAb which was (33.17 mm) which was statically similar to RANAb (31.83 mm). The minimum radial mycelial growth was recorded in NAOAb which was (20.33 mm) which was statistically similar to MYMAb (23.33 mm).

IV. DISCUSSION
A laboratory examination was carried out at Plant Pathology Laboratory of Oil seed Research Center, BARI, Joydevpur, Gazipur to find out morphological and physiological variation among ten different isolates of Alternaria spp. isolated from mustard leaf having typical symptoms of Alternaria blight. Leaves of mustard having typical symptoms were collected from ten different location of Bangladesh and causal organisims were isolated on PDA medium. All the isolates produced light brown to deep brown murifrom conidia with beak. This finding was supported by previous findings [18] . They were also found murifrom conidia which were brownish black. Some researcher worked with Alternaria spp. and found murifrom, obclavate conidia with brownish black [28]. All 10 isolates showed variations in respect of their cultural and morphological characteristics on different media. In respect of cultural characteristics, the isolates of Alternaria spp. showed variation in mycial growth, colony color, shape, textures, subsurface color, zonation conidia production and sporulation time. Remarkable effect of different culture media on radial mycelia growth was observed in Alternaria spp..Significant variation was found in colony color of A. brassicae on PDA medium. Most of the colony color of the isolates were olivacious green to black. The results are partially agreement with [33] who found that the colony color of A. mali isolated from apple was light to dark olivacious with greenish or brownish tinge. In case of A.alternata isolated from ribben plants colony colour black to olivaceous-black or grayish colour on PDA medium was found [24]. Thirty two isolates of A brassicicola for colony color and radial growth were observed by [6]. Colony colour of A. brassicicola varied from olive green to dark olivacious black on PDA. All the isolates of Alternaria spp. colony had circular shaped. The results are in agreement with [38] were identified its morphological and cultural characters of A.brassicae isolates from four different locations, colonies of all the isolates were circular in shape.The colony shape of A.solani isolated from tomato plants were found circular margin with smooth surfaced colony [31]. The entire isolates colony had cottony and velvety texture on PDA medium. The results are in agreement with [3] examined 308 isolates of Alternaria spp. colonies generally had a cottony texture on group 4. Alternaria blotch, causal organism A. mali, colonies varied in their cultural behavior ranging from velvety to cottony [33]. Remarkable variation was observed on spore production and sporulation time on different media and temperature. Potato Carrot Media are found suitable for spore production and sporulation time for maximum isolate followed by CDA and PDA. This result was supported by the [23] found potato carrot broth are suitable for sporulation and spore production A. brassicae. Variation were found in mycial growth, sporulation in different nutrient media like Potato Dextrose Agar, Cauliflower Agar medium and Carrot Potato Agar good for 32 isolates of A. brassicae [33]. Variations were observed in accordance with length, breadth and beck on different isolates of A. brassicae on PDA media. The length of conidia of different isolates varied from 41.56µm to 117.54µm. The breadth of conidia of different isolates varied from 10.34 µm to 23.12 µm. The beck of conidia of different isolates varied from 16.78 µm to 72.65 µm. The horizontal septation varied from 3-7 to 7-11. The vertical station varied from 0-1to 2-3. This result are partially supported with [28] define A. brassicae length of conidia varied from 96 µm -114 µm, breadth varied from 17 µm -24 µm and beck length varied from 45 µm-65 µm and transverse and longitudinal septation varied from 10-11 and 0-6 respectively. 322 isolates of A. brassicae variation was recorded among conidial length, breadth and beak length which range of 51.4-481.2 μm, 6.9-36.0 μm and 16.3 -266.9 μm respectively [16]. Average numbers of horizontal septa were 9.7, vertical septa were 0.8. The horizontal septation of 5 different isolates of A.brassicae varied from 4-13 and vertical from 0-6 [33]. 23 isolates of A. brassicae were collected and found maximum length of conidia ranged from 150 -122 μm with 8 -9 transverse and 2 vertical septation [27] Eight isolates of A. solani were examined [25] and found average conidial size (L×B) was 42.18×15.18 µm and beck size was 13.10µm. In ten isolates of A. macrospora size of conidia ranged from 20.81-56.23 x 9.2-27.10 μm with 1 -6 transverse and 0 -4 longitudinal septa were found [14]. ten isolates collected by [26] of A. alternata the length and width of conidia were varied from 30.99 -42.47 µm and 11.90-17.37 µm respectively. All isolates produced both beaked and unbeaked conidia. The beak length of conidia varied from 18.7-23.81 µm. Alternaria blotch, causal organism A. mali 21 isolates of A. mali were collected from different locations. Average conidial size ranged from 21.36 to 31.74 x 8.34 to 14.48 µm. Among the isolates of A.mali size of conidia 19-50 µm ×5-9 µm in nature and 20-59 µm ×8-13 µm in culture, with 3-8 transverse septa and usually no longitudinal septa or only 1 longitudinal septa were found [33].

V.
CONCLUSION Rapeseed Mustard (Brassica spp.) is the principal oilproducing crop of Bangladesh and Alternaria leaf blight caused by Alternaria brassicae, is one of the major disease of rapeseed mustard. This research was conducted to find out existence of physiological races of Alternaria spp. causing Alternaria leaf blight of mustard on the basis of cultural and morphological aspects. The experiment was laid out in the completely randomized design with three replications. Ten isolates of Alternaria spp. were collected from ten different mustard growing districts of Bangladesh. Three different media and two different temperatures were used to measure growth and development of Alternaria spp.. All the 10 isolates showed variation in the terms of cultural and morphological characteristics. Among three different culture media, potato carrot agar medium at 25˚C showed the best performance in the terms of radial mycelial growth. Colour of the colonies of Alternaria spp. showed variation among ten isolates. Olivacious green to black color colony developed on PDA medium. All the isolates produced Circular colony and the texture were cottony to velvety. All isolates showed compact type compactness. Variation also observed between surface and sub surface colour. Surface colour varied from light brown to deep brown. Subsurface colour varied from light brown to black and pinkish. Zonation was present both surface and subsurface in some isolates and some isolates showed no zonation on both side. Effect of media on sporulation significantly differed among the isolates. The highest number of conidia production was recorded 48.17 to 59.79 × 10 6 /ml was counted RAJAbon Potato Carrot media at 25˚C temperature. Of all the isolates of Alternaria spp. with maximum in isolate RAJAb and minimum in NAOAb. Temperature showed an influence on sporulation. (Data not shown). Effect of media on sporulation time differed significantly among the isolates. The minimum days (4 days) required for sporulation in PCDA followed by CDA. Remarkable variation among different Alternaria spp. isolates were observed in length, breadth and beak size of the conidia. The conidial length varied within a range of 41.56µm to 117.54µm and the breadths were varied from 10.34 µm to 23.12 µm. All isolates were muriform and deep brown to light brown in colour with a beak length of 16.78 µm to 72.65 µm. On the basis of the above results and discussion it can be summarized that-variability exists in the pathogen of alternaria leaf blight caused by Alternaria spp. prevailing in the rapeseed mustard growing areas of Bangladesh. Potato Carrot agar medium and 25˚C temperature were appeared to be the best medium and temperature respectively for the mycelial growth and sporulation of this fungal pathogen. More research should be conducted on molecular characterization of this isolates to find out the phylogenetic relationship. VI.