Diversity of Macrophomina phaseolina ( Tassi ) Goid Based on Chlorate Phenotypes and Pathogenicity

Macrophomina phaseolina (Tassi) Goid causes charcoal disease of oilseed plants. In this study 24 isolates, which were obtained from sunflower, soybean and sesame, were compared based on chlorate phenotypes and pathogenicity tests. For chlorate phenotypes, the isolates were grown on potassium chlorate and stored at 30oС in darkness. For pathogenicity test, seeds of sunflower, soybean and maize plants were placed on 6 – day – old colonies of each Macrophomina isolates grown on PDA and kept at 30oС in the dark. Results indicate that the sesame isolates had more colony radius rate on chlorate minimal medium in comparison to the soybean and sunflower isolates. The sesame isolates were chlorate resistant and grew normally with numerous dark microsclerotia production on the potassium chlorate. The soybean and sunflower isolates were chlorate sensitive and divided into two classes. Class 1, include the isolates that grew sparsely with a feathery like pattern, and the other one had a completely restricted radial growth meaning that M. phaseolina isolates differed in their ability to use certain nitrogenous compounds. Analysis of variance showed a significant difference between the colony radius rates of the isolates at 1% probability level. Based on Duncan’s test, the isolates have been divided in 14 classes. Results of pathogenicity test showed that there was significant difference (P< 0.01) between the isolates. The results confirmed that the feathery like pattern of the isolates was more virulent on soybean and sunflower.


Introduction
Macrophomina phaseolina (Tassi) Goid, is an anamorphic and soil borne fungus with a broad host range that includes 75 plant families and more than 500 species worldwide (Salik, 2007).Many economically significant plants including legumes, vegetables, fruits and fiber crops are attacked by M. phaseolina, a causal agent of charcoal rot disease (Kunwar and Sin, 1986;Sinclair and Backman, 1986;Smith and Carvil, 1997).Estimates of yield reduction due to charcoal rot in the US were 1.98, 0.28, and 0.49 million metric tons in 2003, 2004, and 2005, respectively (Wrather and Koenning, 2006).Macrophomina phaseolina is the most fungal pathogens affecting sunflower in Egypt (Purkayastha et al., 2006).Despite having a wide host range, Macrophomina is a monotypic genus.Efforts to divide M. phaseolina into sub-species were unsuccessful, based on the morphology and pathogenicty, there were extremely intraspecified variations (Dhingra and Sinclair, 1972;Echavez-Badel and Perdomo, 1991).The significant differences of morphological (Mayek-Perez et al., 2001), physiological (Mihali and Taylor, 1995), pathogenic (Mayek-Perez et al., 2001;Su et al., 2001) and genetic (Vandemark et al., 2000;Mayek-Perez et al., 2001;Su et al., 2001;Alvaro et al., 2003;Jana et al., 2003;Aboshosha et al., 2007) diversity have been reported.Control has not yet been achieved through resistance in spite of reports on tolerant genotypes (Smith and Carvil, 1997).Chlorate phenotypes were used as markers for identifying host-specific isolates of M. phaseolina (Das et al., 2006).Many researchers have also found great variability in pathogenicity and morphology among isolates from the same host.It is assumed that during the hyphal fusion, heterokaryosis could occur after mitotic segregation and recombination (Sinclair and Backman, 1986).This may explain the occurrence of cultural types or physiological races of M. phaseolina (Dhingra and Sinclair, 1973;Manici et al., 1995).Recent efforts to classify isolates of M. phaseolina have centered on morphology of the colony on media amended with chlorate (Su et al., 2001).Most fungi can use nitrate as a source of nitrogen.Nitrate uptake does not appear to occur without nitrate metabolism.The metabolic assimilation of nitrate is by reduction to nitrite via nitrate reductase, nitrite is then reduced to ammonia.Chlorite could restrict the growth when the nitrate reductase pathway is active.Unrestricted growth in the sectors resulted from the inactivity of one or more of the five enzymes in the nitrate reductase pathway (Cloud and Rupe, 1988;Mccain and Smith, 1972;Solomonson and Vennesland, 1972).Nitrate reductase also can reduce chlorate to chlorite.The accumulation of chlorite is presumably poisonous to cells.Fungal strains that have functional nitrate reductase are chlorate sensitive, whereas those that are unable to catabolize nitrate are chlorate resistant (Pearson and Leslie, 1987).In this study we have attempted to separate and classify the isolates of M. phaseolina obtained from the oilseed plants on the basis of pathogenicity test, morphology and growth manner on minimal medium containing chlorate.

Fungal isolates
Twenty-four samples were collected from infected stems and roots of soybean, sunflower and sesame plants from Mazandaran Province in northern Iran (Table 1).Each root or stem was thoroughly washed and dried at room temperature.Four small 0.3 cm epidermal sections were excised from each sample and sterilized in 0.8% NaOCl (1min) and washed in sterile water for 1 min.Tissues were placed on potato dextrose agar (PDA) plate followed by incubation at 28±1ºС in darkness for four days.Purification was developed by single microesclerotium culture and maintained on PDA at 28±1ºС (Das, and Fakrudin, 2006).All 24 isolates employed in the present investigation are listed in Table 1.

Phenotypic study
A 1 mm agar plug from the 7 day-old pure culture was placed on minimal medium containing potassium chlorate with some modification (20g agar, 1.6g asparagine, 15g potassium chlorate , 30g sucrose, 2g NaNO 3 , 1g KH 2 PO 4 , 0.5g MgSO 4 •7H 2 O and the final reaction volume was adjusted to 1000 ml with H 2 O) and 0.2ml of trace elements solutions (95ml distilled water, 10g citric acid, 10g ZnSO 4 and 1ml chloroform) was added and kept at 30 ºС in darkness.The pH of the minimal medium was adjusted to 6.5 with KOH before autoclaving (Puhalla and Spieth, 1985).The colony radius rates of the isolates were evaluated by factorial experiment based on completely randomized design (CRD) in four replications.After 48 hours, the colony radius was measured daily with ruler.The minimal medium without potassium chlorate was used as a control treatment.

Pathogenicity test
In this experiment pathogenicity test of isolates was carried out at the seedling stage of soybean (Glycine max L.), sunflower (Helianthus annuus L.) and maize (Zea mays L.) plants in a completely randomized block design.Each treatment (isolate) was replicated three times and included two plates with six seeds per plate.Seeds of the plants were sterilized with 2% sodium hypochlorite for 4 min and rinsed twice in sterile water.Seeds were placed on 6-day-old colonies of the Macrophomina isolate (on PDA plates) and incubated at 30°С in dark condition.Evaluation was done after six days, using the following severity assessment key: 0 = healthy seed; 1 = discoloration of a portion of the seedling in contact with the mycelium; 2 = seed teguments invaded by mycelium and sclerotia but healthy seedling; 3 = seed teguments free from the fungus but seedling infected;4= seed tegument and seedling infected; 5= seed infected and not germinated (Manici et al., 1992).
The disease index was calculated by multiplying the number of seeds by the degree of disease severity.The experimental design was a randomized complete.Analysis of variance (ANOVA) of the data was performed with the MSTAT-C package.

Phenotypic study
Three various growth patterns (feathery spreading growth, restricted growth and dense growth) were observed, when the isolates were grown on the minimal medium containing 120mM potassium chlorate (Table 2, Figure 1).
Restricted and feathery isolates were sensitive to chlorate, whereas dense isolates were resistant to chlorate.Among soybean isolates, feathery isolates were much more abundant than restricted, whereas dense isolates predominated in sesame.Sclerotia production on chlorate medium by the sensitive isolates was too low compared to resistant ones.Isolates of sesame grew more rapidly on the defined medium containing chlorate than did isolates from soybean or sunflower.
All isolates had dense growth when they were grown on the minimal medium without chlorate and could not be differentiated.The growth response of isolates did not alter by increasing chlorate concentrations from 120 to 240 mM, whereas dense and feathery growth appeared similar in the lower concentration (60 mM).Su et al. (2001) and Pearson et al. (1986) reported that mycelial growth of M. phaseolina on chlorate medium was classified into three categories (restricted, feathery and dense).Restricted and feathery isolates were sensitive to chlorate, whereas dense isolates were resistant to chlorate.Manici et al. (1995) reported that four colony chlorate phenotypes were observed.
Analysis of variance of the data showed that the colony radius rate of 24 isolates of Macrophomina phaseolina was significantly different (P< 0.01) on chlorate minimal medium (Table 3).Different colony radius rates were observed in M. phaseolina isolates on chlorate minimal media.Based on Duncan's test, comparison of the means of colony radius rates grouped the 24 isolates in 14 classes on chlorate minimal media (Table 4).Isolate 4 and 7 on chlorate minimal medium had the highest colony radius rate.The chlorate resistant isolates had more radius rates than those in chlorate minimal medium.The results were similar to the reported by Manici (Manici et al., 1992) (Table 4).

Pathogenicity test
The experiment of the pathogenicity test demonstrated that none of the isolates were pathogenic on maize while all isolates showed pathogenic ability on soybean and sunflower (Figure 2).
Analysis of variance showed that the pathogenicity of the 24 isolates of M. phaseolina was significantly different (P< 0.01) on the plant species (Table 5).
Disease indices on sunflower and soybean range between 19 -24 and 27-30 respectively.The means comparison of the different isolates indicate that there was significant difference among isolates on the rate of the disease index.Isolate 2, 5, 11, 23 and 24 indicate maximum disease index (27.00)and were placed in class A, while isolates 10 showed minimum disease index (23.17)and were grouped in class K (Table 6).Femandez et al., (2006) reported that great variability in pathogenicity was recognized among isolates from different host species and between isolates (Femandez et al., 2006).Das et al., (2006) was investigated pathogenicity of some isolates of M. phaseolina that belong to different country.The results showed the pathogenicity of isolates was different and the most aggressive isolates were from Mexico, Brezil and Colombia (Das et al., 2006).
(Table 6) Means comparison of different plants species reaction showed that there was significant difference (P<0.01) on the rate of disease index, hence, the plant species were placed in different groups (Table 7).Soybean with 28.56 had more level of sensitivity (class A) than sunflower (class B) with 22.48.
(Table 7) Mean comparison of interaction between host plants and isolates showed significant difference (P<0.01).All of isolates were pathogenic on soybean and sunflower.Isolates 24, 5, 11 and 23 showed most intensity on both soybean and sunflower, and the intensity of all isolates was more on soybean than on sunflower (Table 8).
The pathogenicity test showed that soybean and sunflower plants are susceptible while maize plant is resistant to Macrophomina.All the 24 isolates that were tested for charcoal rot reaction could infect soybean and sunflower.A broad pathogenic and phenotypic diversity was noticed among Iranian M. phaseolina.Mayek-Prez et al. (2001) studied 84 isolates of M. phaseolina from different geographical regions of Mexico, and identified 43 distinct pathotypes.Su et al. (2001) found high levels of variation in pathogenecity of M. phaseolina.Manici (1995) investigated pathogenecity of M. phaseolina on eight plant species, and all were pathogenic on other plant species except on maize.Isolates were highly virulent on soybean and virulent on sunflower, safflower, sorghum and melon.Studies on M. phaseolina have investigated variations in morphology and pathogenicity among isolates from soybean, common bean and cluster bean (Purkayastha et al., 2006).
In our study the most aggressive isolates originated from North of Mazadaran province were mainly isolated from soybean plants.The study also demonstrated that some chlorate sensitivity in M. phaseolina had some relation with charcoal rot severity in soybean and sunflower.Similar results were reported by Mihali and Taylor (1995) and Mayek-Perez et al. (2001).

Figure 1 .
Figure 1.Owth patterns of Macrophomina phaseolina on a minimal medium containing 120 mM potassium chlorate.A, Feathery, B, restricted and C, dense

Table 1 .
Macrophomina phaseolina isolates characteristics, used in this study

Table 2 .
Chlorate phenotypes of Macrophomina phaseolina isolates obtained from different hosts

Table 3 .
Analysis of variance colony radius rate of 24 isolates of M. phaseolina

Table 4 .
The mean comparison of 24 isolates based on Duncan's test for colony radius rate at minimal medium

Table 5 .
Analysis of variance pathogenicity test of M. phaseolina on the two plant species

Table 6 .
Mean comparison of M. phaseolina pathogenecity on the different plants species

Table 7 .
Pathogenicity of 24 isolates of M. phaseolina on two plant species

Table 8 .
Mean comparison of interaction among different plants species and M. phaseolina isolates