PATHOGENICITY OF Macrophomina SPECIES COLLECTED FROM WEEDS IN COWPEA

Charcoal rot caused by Macrophomina phaseolina is a major cowpea disease causing substantial losses to growers. In the semi-arid region of Brazil, cowpea is one of the most widely used alternatives for crop rotation during the off-season of melon. This favors Macrophomina multiplication because both crops are hosts of this pathogen. The objective of this study was to verify the pathogenicity of Macrophomina phaseolina and M. pseudophaseolina on cowpea. The Macrophomina spp. isolates used were obtained from the roots of Trianthema portulacastrum and Boerhavia diffusa, weed species prevalent in melon production areas in North-east Brazilian. The experiment was carried out in a greenhouse. Cowpea plants cv. „Paulistinha‟ were inoculated with 30 M. phaseolina isolates, 30 M. pseudophaseolina isolates and a reference isolate of M. phaseolina obtained from cowpea roots. All Macrophomina isolates were able to cause disease on cowpea and there were no statistical differences between both Macrophomina species regarding disease incidence and severity. Moreover, 65.2 and 100.0% of the M. phaseolina isolates, and 56.2 and 92.8% of the M. pseudophaseolina isolates, obtained from T. portulacastrum and B. diffusa, respectively, were as severe to cowpea as the M. phaseolina reference isolate from cowpea. These results emphasize the need to establish management practices aiming to control T. portucalastrum and B. diffusa from cowpea production areas, as they can act as potential sources of inoculum and survival for Macrophomina spp.


INTRODUCTION
Walp.) is one of the main legumes cultivated in semi-arid regions of African countries, with 95.9% of world production (7.1 million tons) (FAOSTAT, 2019). However, world production is underestimated because countries such as Brazil and India do not have separate production data between cowpea and common bean (Phaseolus vulgaris L.) (DAMASCENO-SILVA; ROCHA; MENEZES-JÚNIOR, 2016).
In Brazil, cowpea production is concentrated mainly in the North-east and North Regions, with increasing progress in the Midwest region. This crop is considered one of the main protein sources of human food, and an important generator of employment and income in the growing regions (ROCHA et al., 2009;ROCHA;MENEZES-JÚNIOR, 2016). The importance of cowpea is also due to the possibility of being cultivated by small farmers in family farms under irrigated and rainfed conditions, due to its easy management and low production costs, playing an important socioeconomic role (RAMOS et al., 2012;ROCHA;MENEZES-JÚNIOR, 2016).
It is estimated that in the 2016/17 growing season, Brazilian cowpea production reached 789.8 thousand tons in 1.5 million hectares (CONAB, 2019).
Fungal diseases are among the most important limiting factors for cowpea production in Brazil, being responsible for high qualitative and quantitative losses. "Charcoal rot" caused by Macrophomina phaseolina (Tassi) Goid. (RIOS, 1988) is among the most relevant diseases of cowpea crops in Brazil. Due to the thermotolerant condition of this fungus, this disease becomes even more important in the North-east Brazilian Region, as climatic conditions such as high temperature and low humidity favor its development (ATHAYDE SOBRINHO, 2016;GOMES-SILVA et al., 2017;NEGREIROS et al., 2019b).

Macrophomina spp. Isolates
The experiment was carried out in a greenhouse from December 2016 to March 2017 to evaluate the pathogenicity of 61 isolates of two Macrophomina species on cowpea. These isolates were obtained from asymptomatic T. portulacastrum and B. diffusa weed roots collected from commercial melon (Cucumis melo L.) and watermelon (Citrullus lanatus [Thunb.] Matsum & Nakai) production areas in the municipalities of Icapuí (CE), Assú and Mossoró (RN). Crop rotations in these areas include cowpea.

Inoculum preparation
All isolates were placed in potato-dextroseagar (PDA) culture medium and incubated at 28 ± 2 °C in the dark for seven days to be used for inoculum preparation.
Cowpea plants were inoculated following the methodology of the infested toothpick (AMBRÓSIO et al., 2015). Toothpick tips (1.5 cm) were vertically inserted, with the sharp portion of the toothpicks facing upwards, on a filter paper with the same internal diameter as the Petri plate. Subsequently, these plates, properly closed, were autoclaved at 121 °C for 30 min. PDA was then poured into these plates about 4 mm from the end of the toothpicks. After solidification of the culture medium, four 0.5 mm diameter discs with fungal structures (mycelium and sclerotia) were transferred to these plates, equidistantly distributed, and incubated for eight days in an incubator at 30 ± 2 °C, for complete colonization of the toothpicks.

Experimental design and evaluation
Three seeds of cowpea cultivar 'Paulistinha' were sown in plastic pots, with a capacity for 0.75 L, containing commercial substrate Tropstrato HT ® . Then, they were autoclaved twice at 121 °C for one hour, with a 24 h interval in between. Eight days after sowing, thinning was performed to have only one plant per pot. At this moment, toothpicks previously infested with each fungal isolate were inserted into the main stem of the plant at a height of 0.5 cm from ground level. For the uninoculated control, only autoclaved toothpicks were used.
The experimental design was completely randomized, with 61 treatments (representing the isolates) with five replications each, and one uninoculated control. The experimental unit consisted of a plastic pot with a plant. The pots were kept in a greenhouse at an average temperature of 32 °C for 30 days, under natural daylight conditions, and irrigation was manually performed. The experiment was repeated.
The incidence and severity of the Macrophomina isolates were evaluated 30 days after inoculation. Disease incidence was determined by counting plants with symptoms of charcoal rot, and the data were transformed as a percentage (%). To assess the severity of the disease, a scale proposed by Abawi e Pastor-Corrales (1990) was used, with modifications, where 1 = no symptoms; 3 = lesions limited to cotyledonary leaves; 5 = progressing lesions from cotyledonary leaves up to 2 cm in the stem; 7 = extensive lesions, presence of chlorosis and necrosis in leaves and stem; and 9 = presence of pycnidia on stem and plant death.
After the evaluation, all plants were analyzed for fungal isolation to confirm Koch's Postulates. Fragments of the diseased areas were disinfested in a 1.5% sodium hypochlorite solution for 1 min and washed in sterile water. The disinfested fragments were placed in PDA-tetracycline (0.05 g L -1 ) medium and incubated for five days at 30 ± 2 °C.
For each experiment, the preliminary ANOVA was performed to determine whether there were significant differences between the two repetitions of the experiments and whether the data could be combined. Severity and incidence results by isolates of M. phaseolina and M. pseudophaseolina were analyzed with the non-parametric Kruskal-Wallis test at the probability level of 5% (p ≤ 0.05) using the Assistat software, version 7.7 (SILVA; AZEVEDO, 2016). Differences in severity and incidence caused by Macrophomina species were determined using Mann-Whitney test at the 5% significance level using statistix v.9.0 (Analytical Software).

RESULTS AND DISCUSSION
All isolates of both Macrophomina species (M. phaseolina and M. pseudophaseolina) isolated from T. portulacastrum and B. diffusa caused disease on cowpea plants ( Table 2). The results of both experiments were combined because there were no statistical differences between the two experiments (p ≤ 0.05). For both analyzed variables, incidence and disease severity, there were no statistical differences between the Macrophomina species for Mann-Whitney test (p ≤ 0.05) (Figure 1). This fact is quite worrying, given that the production of this crop is prevalent in the Northeast and North regions of Brazil, where temperatures are quite high throughout the year. In fact, M. phaseolina is considered economically important in subtropical regions and tropical countries with a semiarid climate (ATHAYDE SOBRINHO, 2016;GOMES-SILVA et al., 2017;NEGREIROS et al., 2019b), but there is a lack of knowledge about the risks posed by M. pseudophaseolina on cowpea and other crops, because of its recent description (SARR et al., 2014).
In Brazil, M. phaseolina was first reported in 1935, infecting common bean plants in Campinas-SP (BITANCOURT, 1935). In cowpea, under favorable environmental conditions, this fungus can be highly virulent, attacking from the seed to the pod of the plant. Athayde Sobrinho, Viana and Santos (2005) detected the presence of M. phaseolina in 62% of cowpea seed samples analyzed in Brazil. According to Dhingra and Sinclair (1978), infected seeds, symptomatic or asymptomatic, represent the main form of spread of this fungus over long distances.
It is also important to note that 65.2 and 100.0% of the M. phaseolina isolates, and 56.2 and 92.8% of the M. pseudophaseolina isolates, obtained from T. portulacastrum and B. diffusa, respectively, were as severe to cowpea as the M. phaseolina reference isolate from cowpea (Table 2).
Although both Macrophomina species isolated from T. portulacastrum and B. diffusa were pathogenic to cowpea, more than 90% of these isolates, derived from B. diffusa, showed severity in cowpea equal to the M. phaseolina reference isolate. However, the isolates obtained from T. portulacastrum were less severe to cowpea when compared to the isolate from B. diffusa (Table 2).
Weeds are important alternative hosts of plant pathogens (NEGREIROS et al., 2019a;b;SALES JÚNIOR et al., 2019). They can actively contribute to the survival of soil-borne phytopathogenic fungi, especially during the off-season (SALES JÚNIOR et al., 2012).
The coexistence of cowpea with weeds is one of the main factors compromising the development and productivity of this crop. Thus, weeds control is considered one of the main components of the production costs (FONTES; GONÇALVES; MORAIS, 2010). According to Freitas et al. (2009), when uncontrolled, weeds can reduce cowpea grain yield by up to 90%. Moreover, prior knowledge of weed participation as alternative hosts of soil-borne phytopathogenic fungi may be of great relevance in the management of root diseases, especially in the off-season, as they may play an important role in disease epidemiology (SALES JÚNIOR et al., 2012).
The weeds species T. portulacastrum and B. diffusa occur frequently in the main areas of melon (SALES JÚNIOR et al., 2019), watermelon (SILVA et al., 2013), and corn (Zea mays L.) (NASCIMENTO et al., 2011) cultivation in the state of RN, Brazil. In addition to Macrophomina spp., they have also been reported to host, Monosporascus spp. and Rhizoctonia solani Kühn, which are soil-borne pathogens associated with root rot and vine decline on melon crops (SALES JÚNIOR et al., 2012;NEGREIROS et al., 2019a).
In Brazil, there are no chemicals registered for field control of this pathogen in any crop, and only products for seed treatment are available (AGROFIT, 2019). Trianthema portulacastrum and B. diffusa, even with no visible symptoms in the plants, when present in a cowpea production area can compete for water, nutrients and light, and also act as a source of inoculum for M. phaseolina and M. pseudophaseolina, which may cause charcoal rot in the plants and, consequently, a reduction in yield.

CONCLUSIONS
Our results emphasize the need to establish management practices aiming to control T. portucalastrum and B. diffusa from cowpea production areas, as they can act as potential sources of inoculum and survival for Macrophomina spp.