Cultural, Morphological and Pathogenic Variability in Different Isolates of Alternaria carthami of Safflower from Different Geographical Regions of Maharashtra

Cultural, morphological and pathogenic studies revealed a wide range of variability of among the 20 isolates of A. carthami of safflower which represented four geographic regions of the state of Maharashtra. All 20 test isolates exhibited a great cultural variability in mycelial growth, colony colour, growth speed, colony shape and sporulation. Significantly highest mycelial growth was found in isolate AcHl (90.00 mm), followed by AcBl (89.67 mm) and AcAn (88.83 mm). Among morphological variability in respect of mycelial width, conidial dimensions, beak length and number of vertical and horizontal septa eight isolates exhibited large mycelial width (7.05 to 9.80 μm), six isolates with medium sized with mycelium width (5.40 to 6.46 μm) and six with small sized mycelium width (3.25 to 4.86 μm). On the basis of average conidial dimensions (length X breadth), the test isolates were categorized into three groups viz., large, medium and small sized conidia were found in nine isolates of which average length ranged from 36.37 to 50.32 μm and width from 13.06 to 17.19 μm; seven isolates were with medium sized conidia (26.80-35.37 X 9.94-14.73 μm) and rest four isolates with small sized conidial (22.45-25.17 X 7.28-9.27 μm). About eight isolates exhibited long beak length (11.05 to 16.43 μm), seven isolates with medium beak length (8.13 to 11.27 μm) and rest five isolates were of short beak length (6.04 to 7.46 μm). Among the test isolates, horizontal septation on conidia was ranged from 1 to 12 and vertical septation from 0 to 3. All of the 20 isolates of A. carthami exhibited a wide range of pathogenic variability. However, the aggressive isolates (viz., AcHl, AcBl, AcAn, AcJg, etc.) showed least incubation period, highest leaf spot frequency with maximum sized leaf spots.

Safflower is the most important rabi oilseed crops grown in India.In general, the crops have low average productivity due to the prevalence of various biotic and abiotic stresses.Diseases are among the major biotic stresses.Alternaria blight caused by Alternaria carthami is an important and a widespread destructive disease of safflower.Depending on the severity of the disease, yield losses as high as 25-60% have been reported in India (Singh and Prasad, 2005).
The Alternaria spp.are one of the widely distributed phytopathogens infecting a wide range of agronomical and horticulture crop plant species, cultivated worldwide.In Alternaria spp., the existence of a high level of variability viz., cultural, morphological and pathogenic etc. have earlier been reported by several workers (Rajender et al., 2013;Sharma et al., 2013;Giri et al., 2014;Nikam et al., 2015).The variants within population of the phytopathogen may certainly affect the rate of disease development and can induce infections even in disease tolerant or resistant host plant species.To develop an effective programme of breeding for disease resistance comprehensive understandings of pathogenic, morphological, cultural and pathogenic variability are essential.Therefore, in present studies, cultural, morphological and pathogenic variability of A. carthami isolates collected from various agroclimatic zones and geographical regions of state of Maharashtra was attempted.

Cultural, morphological and pathogenic variability among A. carthami isolates Cultural variability among A. carthami isolates
Twenty test isolates of A. carthami obtained one each from twenty districts under various geographical regions of the Maharashtra state were aseptically inoculated on PDA medium separately, maintaining three plates / isolate and incubated at 28±2 0 C, in an BOD incubator.Observations on cultural characteristics viz., colony diameter, colony colour / pigmentation, appearance, growth rate, shape, margin and zonation were recorded after a week of incubation and sporulation was recorded at 15 days of incubation.

Morphological variability among A. carthami isolates
The morphological characteristics viz., hyphal width, size of conidia, length of beak of each test isolate (10 days old pure culture growth on PDA) were recorded by measuring with ocular micrometer, which was calibrated using stage micrometer, by applying standard procedure given by Aneja (2001) under the compound microscope (make: Labomed Vision 2000) at 400x magnification and under 10 random microscopic fields.Similarly, numbers of transverse and longitudinal septa / conidium were counted.

Pathogenicity assay and pathogenic variability among of A. carthami isolates
In order to confirm identification of the disease and its causal agent, the pathogenicity test was attempted in pot culture under screen house conditions.Seeds of safflower cv.Manjira susceptible to Alternaria blight (A.carthami) were surface sterilized with 0.1 % HgCl2 and sown (@ 10 seeds/pot) in the earthen pots (30 cm dia.) filled with steam sterilized potting mixture of soil : sand : FYM (2 : 1 : 1).After two week, two healthy growing safflower seedlings per pot were maintained, watered regularly and kept in the screen house for further growth and development.The spore-cummycelial suspensions of A. carthami test isolates was prepared separately from 10 days pure culture in plates, by flooding with 5-10 ml sterile distilled water.This resultant suspension was suitably diluted with sterile distilled water to get inoculum concentration of 2 x 106 spores/ml.Thirty days old seedlings (2 / pot/ isolate / replication) of safflower cv.Manjira growing in earthen pots were artificially spray inoculated the spore-cum-mycelial suspension separately of the test isolates (Giri et al., 2013), the experiment was planned with CRD and all the test isolates replicated thrice.Safflower cv.Manjira seedlings grown in earthen pots and sprayed with sterile water (without inoculum) were maintained as uninoculated suitable control.These pots (both inoculated and uninoculated) were covered with polythene bags during evening hours and kept overnight, watered regularly to create optimum relative humidity and maintained in screen house for development of the disease symptoms.
From the artificially inoculated and Alternaria blight diseases safflower leaves the test isolates were reisolated separately on PDA medium and incubated at 28±2 0C.After a week of incubation the cultural and morphological characteristics developed of the test isolates were observed and compared with the characteristics (cultural and morphological) of the original test isolates obtained from naturally Alternaria blight diseased safflower foliage.To satisfy Koch's postulates, symptoms developed on artificial inoculated safflower leaves were compared with original symptoms on naturally diseased plants.Observations on incubation period (days to expression of initial symptoms), number of lesions / plant and diameter of the lesions were recorded.

Cultural variability among A. carthami isolates
The results obtained on cultural characteristics viz., colony / mycelial growth, growth rate, colony appearance, margin, sporulation and concentric zonation etc in respect of 20 test isolates of A. carthami grown on PDA are presented in Table 1 and depicted in Plate 1.

Colony colour
On the basis of colony colour, the test isolates were categorized into eight groups.Group I consisted four isolates with creamy white colony (AcPb, AcOs, AcSl and AcJg), group II contained of four isolates with creamy black colony (AcNd, AcAb, AcAn and AcDl), group III consisted two isolates with off white colony (AcHl and AcSt), group IV consisted two isolates with grayish black colony (AcBd and AcYt), group V consisted two isolates with light gray colony (AcJl and AcNs), group VI consisted three isolates with brown colony (AcWs, AcAk and AcBl), group VII consisted two isolates with olivaceous black colony (AcLt and AcNb) and group VIII consisted a single isolate with yellowish colony (AcAm).

Colony growth rate
On the basis colony growth rate, the test isolates were categorized as fast growing, moderate growing and slow growing.In fast growing category, the seven isolates included were AcPb, AcNd, AcHl, AcWs, AcAn, AcBl and AcJg; medium / moderate growing category the six isolates included were AcSt, AcOs, AbBd, AcAb, AcAk and AcLt and in slow growing category the seven isolates included were AcJl, AcYt, AcDl, AcAm, AcNb, AcNs and AcSl.

Colony shape and margin
On the basis of colony shape (circular or irregular) and colony margin (smooth or rough), the test isolates were categorized into two groups.

Morphological variability among A. carthami isolates
Results (Table 2 and Plate 2) revealed that all the 20 test isolates of A. carthami exhibited a wide range of variability in respect of mycelial width, conidial dimensions, beak length and number of vertical and horizontal septa.

Mycelial width
On the basis of mycelial width, the test isolates were categorized into three groups viz., large, medium and small sized mycelium.About eight isolates exhibited large mycelial width in the range of 7.05 to 9.80 µm.However, it was maximum in the isolate AcHl (9.80 µm), followed by AcBl (9.25 µm), AcAn (8.46 µm) and AcJg (8.15 µm); while, in rest of the four isolates (AcPb, AcWs, AcNd and AcOs) the mycelial width was ranged from 7.05 to 7.81µm.
In small sized mycelium category, about six isolates were grouped with mycelial width in the range of 3.25 to 4.86 µm.However, it was maximum in the isolate AcJl (4.86 µm), followed by

Conidial size
The results revealed that the test isolates, exhibited a wide range of variability in respects of average conidial dimensions (length X breadth) and on the basis of which they were categorized into three groups viz., large, medium and small sized conidia.
In large sized conidial group, about nine isolates were included with their average conidial length in the range of 36.37 to 50.32 µm and their average width in the range of 13.06 to 17.19 µm.Among these isolates, highest average conidial size (length X breadth) was recorded in the isolate AcAk (50.32 X 15.02 µm), followed by the isolates viz., AcLt (45.In the medium sized conidial group, seven isolates were included, of which average conidial size was ranged from 26.80-35.37X 9.94-14.73µm.Among these isolates, maximum average conidial size was recorded in the isolate AcAm (35.37 X 9.94 µm), followed by the isolates viz., AcJl (32.27 X 12.76 µm), AcYt (30.00 X 12.27 µm) and AcNd (29.38 X 11.24 µm).Rest of the three isolates in this group recorded average conidial size in the rage of 26.80-28.20X 10.78-14.30µm.

Beak length
On the basis of conidial average beak length, the test isolates were also categorized into three groups viz., long, medium and short beaked.
Thus, from this foregoing results it has been inferred that all 20 test isolates of A. carthami representing four geographic regions of the Maharashtra state exhibited a varied range morphological characteristics.The mycelial width, conidium size and their beak length were varied from small to large, as well as septations (H and V) were ranged from 1-12 (horizontal) to 0-3 (vertical).
Cultural and morphological variability studies in A. carthami, A. brassicae, A. sesame, A. solani, A. lini, isolates was attempted earlier by several workers.Mortensen (1983) reported mycelial growth of the A. carthami isolates ranged from 81 to 32 mm on PDA within 8 days, conidial amount from 0.1 X 10 5 to 19.3 X 10 5 per plate.Mycelial growth in isolate 4 and 43-1 was rapid mycelial growth (81 and 79 mm) and it was slow isolates S-a, 22 and 49-1 (65, 64 and 60 mm, respectively).Vishwanath et al. (2002) reported cultural variability among three isolates of A. brassicae in respect of growth and colony chararacters.Savitha and Naik (2004) that the isolates of Alternaria spp.infecting sesamum produced fluffy to raised dark brown or light grey mycelium (72-88 mm) on PDA.Cultural variability among the isolates of various Alternaria spp. reported earlier were A. brassicae (Prasad et al., 2012;Singh and Singh, 2014), A. helianthi (Rajender et al., 2013).

Pathogenicity test and pathogenic variability
Pathogenicity test (Plate 3) of A. carthami (20) was conducted in screen house using Alternaria blight susceptible safflower cv.Manjira and results obtained on pathogenic traits viz., incubation period, number of spots and size of the spots are presented in Table 3.

Symptoms
All 20 test isolates of A. carthami were found pathogenic to safflower.The symptoms induced under pathogenicity test were identical to those symptoms observed on naturally diseased safflower crop foliage during survey.

Incubation period
Results revealed that among various pathogenic traits of A. carthami, the incubation period was varied in the susceptible safflower cv.Manjira.On the basis of incubation period (day to expression of first symptom), the test isolates were categorized in three groups viz., A (highly aggressive with minimum of 5-6 days incubation period), B (moderately aggressive with moderate of 7-8 days incubation period) and C (less aggressive with 9 or more days incubation period).Accordingly, in group A the isolates (04) included were AcHl, AcAn, AcBl and AcJg; in group B the isolates (12) included were AcPb, AcNd, AcSt, AcOs, AcAb, AcWs, AcAk, AcLt, AcBd, AcJl, AcYt and AcSl and in group C the isolates (04) included were viz., AcAm, AcNb, AcDl and AcNs.

Size of leaf spots
Size / diameter of the spots induced by A. carthami isolates on foliage of safflower cv.Manjira was also found to be varied significantly among the test isolates and it was ranged from 2.80 to 17.14 mm 2 .
Thus, from this ongoing results it was concluded that all 20 isolates of A. carthami representing four geographic regions of the Maharashtra state exhibited a wide range of pathogenic variability.However, the aggressive isolates (viz., AcHl, AcBl, AcAn, AcJg, AvNd, etc.) showed highly virulent least incubation period, highest leaf spot frequency and maximum sized leaf spots.Whereas, moderately virulent / aggressive isolates exhibited each moderate incubation period, leaf spot frequency and their size.In less virulent isolates viz., AcAm, AcDl, AcNb and AcNs through incubation period was maximum (> 9 days), but leaf spot frequency and their size were of lower degree.
Pathogenic association of A. carthami with safflower causing Alternaria leaf spot / blight was reported earlier by several workers under controlled conditions of screen house by inoculating spore cum mycelial suspension (A.carthami) on susceptible safflower cultivars (Singh and Prasad, 2005 reported existence of pathogenic variability in A. macrospora isolates from cotton.They found that in virulent isolates the incubation period very short (8-9 days), large lesion size and maximum number of lesions per unit leaf area; whereas, the less virulent isolates required maximum incubation period (8-13 days), small sized lesion and their minimum frequency.
Thus, in present study pathological, cultural, morphological and pathogenic variability observed among the isolates of A. carthami may be attributed to their geographic distribution in the Maharashtra state, long tern influence of weather parameters at a particular location, ability of the pathogen to adopt the safflower varieties grown etc.

Table 1 .
Cultural variability among the isolates of A. carthami

Table 2 .
Morphological variability among the isolates of A. carthami H : Horizontal, V : Vertical

Table 3 .
Pathogenic variability among A. carthami isolates * : Mean of three replications, Av. : Average, No. : Number Plate 1 : Cultural variability among the test isolates of A. carthami Plate 2 : Close-up view of conidia of A. carthami test isolates Plate 3 : Pathogenicity test and pathogenic variability of A. carthami isolates on safflower Cv.Manjira ; Ranaware et al., 2010; Gholve et al., 2015).The cultural and morphological characteristics of A. carthami found in present study are in consonance with the earlier reported (Deokar and Raghuwanshi, 2002; Taware et al., 2014).Gholve et al. (2015) reported that A. carthami produced initially white, cottony profused aerial mycelium, which gradually turned greenish grey in colour.Aged culture completely black with no aerial mycelium on Potato dextrose agar Microscopic characteristics such as brownish black septate mycelium and dark brown beaked conidia with transverse and longitudinal septation of A. carthami observed during present study were also reported earlier by several workers (Ranaware et al., 2010; Prasad et al., 2012; and Gholve et al., 2015).Pathogenic diversity among isolates of the Alternaria spp.infecting various oilseeds and vegetable crops was reported in past.Meena et al. (2011) reported pathogenic variability in A. brassicae isolates in respect of disease severity on host differentials and lesion size.Virulent isolates produced large sized lesions with higher per cent disease severity.Sigh et al. (2013), Sharma et al. (2013) and Giri et al. (2014) were of similar opinion in respect of pathogenic variability in A. brassicae causing leaf blight of rapeseed-mustard.Kolte et al. (2005) reported pathogenic variability in leaf spots produced.Virulent isolates produced large spots with dark margins.Jadhav et al. (2011)