Is the recently described Macrophomina pseudophaseolina pathogenically different from Macrophomina phaseolina ?

1 Centre Régional AGRHYMET, Département Formation et Recherche, BP 11011 Niamey, Niger. 2 Centre National de Recherches Agronomiques, Laboratoire de phytopathologie ISRA/CNRA.BP: 53 Bambey, Sénégal. 3 Centre Régional d‟Etude pour l‟Amélioration de l‟Adaptation à la Sécheresse, BP 3320 Thiès, Sénégal. 4 Université Cheikh Anta Diop/Institut de Recherche pour le développement, BP 1386 CP 18524, Dakar, Sénégal.


INTRODUCTION
Cowpea and groundnut production is a significant economic activity in Senegal.Both crops are increasingly damaged by charcoal rot caused by Macrophomina phaseolina, which is supposed to be due to increased conditions of drought (Koenning and Wrather, 2010).In the Sahelian zone of West Africa, charcoal rot is estimated to cause a yield loss of 10%, equaling 30,000 tons of cowpea worth approximately US$ 146 million only for Niger and Senegal (Ndiaye, 2007).M. phaseolina is a soil-and seed-borne polyphagous pathogen causing various rots and blights in more than 500 crop species (Sinclair and Backman, 1989;Dhingra and Sinclair, 1977).
Since about 1987, charcoal rot is in the Sahel recognized as the most important diseases of legumes, including cowpea and groundnut (Paré, 1990;Adam, 1995;Gaïkwad and Sokhy, 1987).Characteristic disease symptoms include presence of black sclerotia on the lower part of the stem and wilting and drying of the leaves and subsequently the whole plant at the flowering and fruiting stage.M. phaseolina can also infect roots which show necrotic lesions, leading to pre-or post-emergence seedling damping off or bad plant growth (Bouhot, 1967;Adam, 1986).The disease development was reported to be affected by host growth stage and environment (Csöndes et al., 2007).High root infection was associated with hot and dry weather occurring early in the growing season (Cloud and Rupe, 1994).Furthermore Manici et al. (1995) reported that in Italy, isolates from cold area grew better at low temperature and showed a better adaptability to 40°C.Whereas, isolates from the Mediterranean climate grew fast at high temperature (30, 35 and 40°C) but showed the poorest adaptability at low temperature.In the Sahel, the 1970-1990 period is characterized by frequent drought spells during the period of crop growth and the temperatures during the hottest months of the dry season are high (30-45°C) (Morrel, 1992).The results of a study on the influence of temperature and soil humidity on the germination of microsclerotia of M. phaseolina in a sandy soil showed that the greatest percent of germination occurred at 30 and 33°C at -80, -300 and -1,500 kPa water tensions (Viana and Souza, 2002).
Recently, based on 189 isolates which were morphologically identified as M. phaseolina, Sarr et al. (2014) identified two well-defined clades on the basis of a multi gene DNA analysis of five loci.This led to the description of a novel Macrophomina species, Macrophomina pseudophaseolina Crous, Sarr & Ndiaye (Sarr et al., 2014).Up to now M. pseudophaseolina species is known only from Senegal where it has been isolated from Abelmoschus esculentus, Arachis hypogaea, Hibiscus sabdarifa and Vigna unguiculata and some time in a joint infection with M. phaseolina.Although the morphology of M. pseudophaseolina appears to be largely similar to that of M. phaseolina, its ecological attributes need further study.
Therefore, this study aimed to investigate the pathogenicity of M. pseudophaseolina on three varieties of cowpea under two temperature regimes.

Collection of Macrophomina spp. isolates
From September to November 2011, charcoal rot-affected stems or roots of cowpea (Vigna unguiculata), groundnut (Arachis hypogaea), sorrel (Hibiscus sabdarifa), sorghum (Sorghum bicolor) and okra (Abelmoschus esculentus) were collected from the major production areas of Senegal.The sampled plant tissues were washed with tap water, cut into small pieces, surface-sterilized in 0.8% NaOCl for 1 min, blotted dry with paper towels, placed in a paper bags and dried in an oven at 37°C for 7 days.Dried tissues were ground in a mixer mill (Retsch, GmbH and Co. Type MM2) for 4 min at 600 rotations min -1 and sieved through 180 and 45 µm screens.50 mg from each sample were mixed with 100 ml of PDA amended with 5 mg chloramphenicol and 225 mg PCNB, and plated as described by Ndiaye et al. (2007).From each sample, 1-3 colonies were transferred separately after 5-7 days incubation at 33°C in a fresh PDA medium.Each colony was then considered as an isolate.Isolates of Macrophomina were made at the CILSS/AGRHYMET Phytopathology Laboratory in Niamey, Niger and they were genetically characterized at the CBS-KNAW Fungal Biodiversity Centre (Utrecht, The Netherlands).Details regarding the geographical and host origin and Macrophomina species of the isolates used in this study are listed in Table 1.

Bioassay preparation
Sterilized soil amended with organic fertilizer (N-P-K, 1.5-1-1) was used to fill sterilized 0.5-L pots.After moistening the soil, three 2-cm deep planting holes were made at the pot surface.A 5-mm diameter disc was cut out from the margin of a 3-day-old fungal culture growing on PDA and one disc was placed in each hole.The discs were slightly covered with soil and a surface-disinfested (2.5% NaOCl for 5 min) seed was added, which again was covered with soil.

Climatic chamber experiments
The bioassay was carried out in a "SANYO Versatile Environmental Test chamber" model MLR -351 in a complete randomized design block with two factors (cowpea varieties (3) and Macrophomina species (3 treatments: M. phaseolina, M. pseudophaseolina and a non-inoculated control) with three repetitions per treatment combination.The pots were placed on trays in climatic chambers and plants grown at 34 and 24°C (climatic chamber temperature 1) alternatively for 12 h and at 36 and 26°C (climatic chamber temperature 2).These growing temperatures correspond to the mean maximal and minimal temperature for the period 1950-1959 (humid period) and 2000-2009 (dry period) in the Sahel, respectively (Alhassane et al., 2013).The lightening was insured by 15 20-mv-lamps, arranged along the 3 doors of the climatic chamber.The RH levels recorded were 90 ± 10% and 70 ± 20% for the climatic chamber temperatures 1 and 2, respectively.
The plants were watered two times and the number of stem necrosis, withered and dead plants was recorded weekly for the incidence determination.For primary inoculum potential (total number of microsclerotia produced by plants of a pot) determination, the plants were uprooted 45 days after sowing.The root systems were washed with tap water, blotted dry with a paper towel and the roots and stems were air-dried in an oven at 37°C.Subsequently, the dried plants were weighed and milled, 150 mg of the powder was mixed with 100 ml of SS-medium and poured in 10 Petri dishes (Ndiaye et al., 2007).The plates were incubated at 30°C for 10 days, the number of germinated microsclerotia were counted.

Data analysis
Data were subjected to analysis of variance with Genstat® for Windows 12 th Edition (IACR-Rothamsted, Harpenden, Hertfordshire, UK).Treatment means were separated by the Ducan"s multiple range test (DMRT).To compare Macrophomina species in the different climatic chamber temperatures, an analysis was done on the collected data with a multifactorial design of 2x2x3 with 3 replicas where the factors are: Factor A: Climatic chamber temperature (34/24 and 36/26°C); Factor B: Macrophomina species (M.phaseolina and M. pseudophaseolina); Factors C: Cowpea variety (cvs.Apagbaala, IT93K-503-1 and Mouride).

Plant dry weight
The plant dry weight was only affected significantly by the growing temperature and the cowpea varieties (Table 2).It was higher at 34/24°C than at 36/26°C and at this last temperature, cv.Apagbaala produced lesser dry biomass than the others cowpea varieties (Figure 4).

Potential primary inoculum
The potential primary inoculum was estimated as the number of microsclerotia produced by plants of each treatment unit.It was significantly low in cvs.Apagbaala and IT93K-501-1 as compared to cv.Mouride in both climatic chamber temperatures.However, the mean potential primary inoculum was higher at 34/24 than at 36/26°C (Figure 5).

DISCUSSION
The recent discovery on M. pseudophaseolina raises questions about its pathogenicity which is relative to M. phaseolina.Here, for the first time, the pathogenicity to three varieties of cowpea at two temperature regimes were compared.In general, differences between the two Macrophomina species were limited.The interactions Macrophomina species × growth temperature and Macrophomina species × cowpea varieties were however significant for the disease incidence and intensity as microsclerotia -1 g tissues (Table 2).In cv.Mouride, M. pseudophaseolina induced higher incidence and more microsclerotia g -1 tissue production at 36/26°C than M. phaseolina (Figures 2 and 3), but induced less disease development in Apabaala.
Temperature strongly affected the incidence and the density of microsclerotia in plant tissue being higher at 36/26°C than at 34/24°C.The observed difference in disease incidence may be due to the variation in pot soil humidity more which is pronounced at 36°C than the direct effect of the temperature on the pathogen.Indeed, pots were watered once every four days, and the 4°C higher growth conditions in the climatic chamber temperature 2 lead to a more rapid reduction of the pot soil moisture suitable for charcoal rot symptom expression (Meyer et al., 1974;Ali and Ghaffar, 1991;Sheikh and Ghaffar, 1979;Kending et al., 2000).Such effects of temperature on Macrophomina development have been reported repeatedly (Odvody and Dunkle, 1979;Mihail, 1989;Iqbal and Mukhtar, 2014) and cowpea varieties (Ndiaye, 2007) on charcoal rot development.
Among the cowpea varieties, cv.Mouride showed the highest incidence and density of microsclerotia in plant tissue in both growth temperatures.These results supported the field observations reported by ISRA (data no published) but also raised the question relative to its role in the rapid progress of the charcoal rot disease.Indeed cv.Mouride is one of the largest distributed varieties in the cowpea production zone of Senegal (Louga, Thiès and Diourbel), where it was introduced as response to the shortening and variation of the rainfall.The variety is resistant to drought, cowpea viruses and bacterial blight (Cisse al., 1995).Despite these evident positive characteristics of this cowpea variety, planting in Macrophomina affected areas should be discouraged.
In spite of the small differences in pathogenicity between M. phaseolina and M. pseudophaseolina, the latter Macrophomina species seems somewhat more aggressive in the susceptible variety (more microsclerotia/g tissue and less biomass production in the susceptible variety) at the higher temperature tested (36/26°C).This effect is however probably of lesser importance for management of the disease.So, based on the current results, it is phytopathologically not important to discern between the two Macrophomina species for charcoal rot management in cowpea.More pathogenicity studies on other host crops should be run in order to see if there is a preference for other hosts and to understand why M. pseudophaseolina is less distributed than M. phaseolina (less than 1% in 180 isolates collected from Senegal and Niger) (Sarr et al., 2014).

Figure 1 .Figure 2 .Figure 3 .
Figure 1.Plant stand of 3 cowpea varieties one week after planting in artificially infested soil and growing in a climatic chamber at 34/24 and 36/26°C.Bars = Standard error of means.

Figure 4 .Figure 5 .
Figure 4. Effect of cowpea varieties as affected by growing temperature on the plant dry weight, 45 days after planting and growing in a climatic chamber.Bars = Standard error of means.

Table 1 .
Isolates of Macrophomina phaseolina (Mp) and M. pseudophaseolina (Mps) collected from different crops and locations in the main production areas of Senegal used in this study.
*Corresponding author.E-mail: M.Ndiaye@agrhymet.Tel: 0022796597900.Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License

Table 2 .
ANOVA table of a multiple experiments for plant stand, charcoal rot incidence, tissue population density of Macrophomina, plant dry weigh and potential primary inoculum 45 days after planting cowpea.