Isolation and characterization of Macrophomina phaseolina isolates prevailing in Sindh , Pakistan

Macrophomina phaseolina (Tassi) Goid is economically important fungal pathogen with wide host range. In this study we found morphological and phenotypical variation among isolates of the fungus infecting sunflower crop in Sindh province of Pakistan. Thirty two M. phaseolina isolates were obtained from infected plant samples of sunflower collected from different ten districts viz., Badin, Thatta, Hyderabad, Tando Muhammad Khan, Shaheed Benazirabad, Mirpurkhas, Sanghar, Dadu, Sukkur and Khairpur. Significant variation in characteristics was noticed among collected isolates. They were usually black, blackish-gray, grayish-black and gray in colony color. Generally three growths pattern; dense, feathery and restricted were seem. Among them dense was most common (50.0%) followed by feathery (34.36%) and restricted (15.64%). Maximum colony growth (90.0%) and minimum (65.0%) was recorded after 7 days of inoculation on medium. Feathery growth was generally fast and restricted growth was slow. Maximum average linear colony growth (13 mm) and minimum (9 mm) growth was noted. Isolates showed significantly varied sizes of microsclerotia. Maximum sclerotia size (124.0 μm) was obtained from fields of Badin while, minimum size of microsclerotia (83.0 μm) from sunflower fields of Sukkur. Microsclerotia from dense growth pattern were black and big in size and small microsclerotia from gray growth pattern.


Collection of samples
Plant samples showing charcoal rot symptoms and soil samples near root zone of infected plants were collected from various farmer's fields and research stations at sunflower growing districts of Sindh.All samples were brought to the laboratory for further studies.

Macrophomina phaseolina isolates Plant sample
After surface washing of plant samples under tap water; small pieces (0.5 cm) from symptomatic stem and roots were cut and disinfected with 1% sodium hypochlorite for 2 minutes and then rinsed three times in sterilized distilled water.Infected small pieces were placed on Petri plates (5pieces per plate) containing potato dextrose agar (PDA) medium.All Petri plates were incubated at 30 o C for 7 days.Further, pure culture of the fungus was maintained by hyphal tip method.Isolates of M. phaseolina were named in abbreviate along with host plant and where numbered in numerical digits starting from 1 to 32.

Macrophomina phaseolina isolates Colony growth
To calculate growth rate of isolates, 5 mm disc from active growth of fungus was taken and placed at center of 90 mm diameter Petri plate containing sterilized PDA medium.All Petri plates were incubated at 30 o C for 7 days.Each treatment was replicated five times.Observation on growth speed was taken by measuring diameter of Petri plates in mm per day and at the time of experimental termination.Furthermore observations on colony color and growth pattern were recorded for each isolates.Sclerotia size Slides were prepared from 7 days old pure culture of each isolates and examined under microscope ocular micrometer.Length and width of ten randomly selected sclerotia was measured in µm and were presented as average of both dimension.Additionally shape of sclerotium was noticed for each isolate.

Statistical analysis
Data regarding radial growth, average growth (mm/day) and sclerotia size (µm) was statistically analysis for determining LSD at α = 0.005 using STATISTIX v. 8.1 software (Analytical Software).

Results
Remarkable variation in morphological and phenotypical characteristics was noticed in purified cultures of M. phaseolina.Thirty two M. phaseolina isolates were obtained from infected plant samples of sunflower collected from different ten districts of Sindh (Table 1 & Figure 1).They were usually black, blackish-gray, grayish-black and gray in colony color (Table 2).Generally three growth patterns; dense, feathery and restricted were seen (Table 2).Among them dense was most common (50.0%) followed by feathery (34.36%) and restricted (15.64%) (Table 2).Maximum colony growth (90.0%) and minimum (65.0%) was recorded after 7 days of inoculation on PDA medium.Feathery growth was generally faster than restricted type growth (Table 2).Average linear colony growth per/day ranged from 9-13 mm, which significantly differ with each other (Table 2).Sclerotium size also significantly varied among the isolates.Isolates showed significantly varied sizes of microsclerotia.MPS16 isolate collected from fields of Badin showed maximum sclerotium size (124.0µm) while, minimum size of sclerotium (83.0 µm) was measured in MPS29 obtained from sunflower fields of Sukkur (Table 2).Microsclerotia from dense growth pattern were black and big in size and small microsclerotia from gray growth pattern.

Table 1 . Isolates of Macrophomina phaseolina obtained from different sunflower farmer's fields of Sindh
Saleh et al. [9]and Atiq et al.[10] also reported three types of colony growth (dense, feathery and restricted) patterns of M.