IDENTIFICATION OF SOURCES OF RESISTANCE AGAINST CHARCOAL ROT IN COWPEA 1

- Cowpea [ Vigna unguiculata (L.) Walp.] is an important socioeconomic crop in Brazil, mainly in the Northeast and more recently in the Midwest of Brazil. Charcoal rot caused by Macrophomina phaseolina (Tassi) Goid, is an important disease in semiarid regions, where edaphoclimatic conditions are favorable to the development of disease. The aim of this study was to evaluate the response of 100 cowpea lines to two isolates of M. phaseolina . The experiments were conducted in a completely randomized design, with five replications (two plants per pot). The main variables evaluated were lesion length and relative growth compared to control (RGCC). Among the evaluated accessions, 15% of the lines were resistant to isolate 59 and 11% of the lines were resistant to isolate CMM 2106 of M. phaseolina . Therefore, these accessions can be used as a source of resistance to M. phaseolina by farmers directly as new cultivars or in future hybridizations of cowpea genetic breeding programs.


INTRODUCTION
Walp.] has great socio-economic importance to Brazil, as it contributes to the generation of jobs and income for thousands of people, especially for family farms. The world production of cowpea reaches 7,233,408 tons in an area of 12,496,305 ha. Nigeria is the largest producer, with 2,577,393.32 tons (FAO, 2018;BOUKAR et al., 2018). Brazil has an area of 1,352,500 ha, a productivity of 494 kg ha -1 , and a total production of 668,000 tons of cowpea (CONAB, 2021). The largest national production is in the state of Mato Grosso, followed by the states of Ceará, Bahia, and Piauí (CONAB, 2021). The low productivity of cowpea crop in Brazil is owed to the non-use of technologies and inputs for production because the non-use of certified seeds, inadequate cultural treatments, and occurrence of pests and diseases affect crops.
Charcoal rot is among the main diseases that affect cowpea crops, and it is caused by the fungus Macrophomina phaseolina. The fungus is a soilborne pathogen that attacks the root system of several host plants and infects their vascular bundles, making translocation of nutrients and water difficult. The fungus has resistance structures known as micro -sclerotia, which remains after long periods of crop rotation, and once they are introduced into a cultivated area, they are hardly eradicated (CORREIA; MICHEREFF, 2018).
Few sources of resistance to M. phaseolina have been identified in cowpea genotypes. Muchero et al. (2011) evaluated 14 genotypes evaluated under moderate water stress conditions, and there were different genotypic responses to M. phaseolina infection. The genotypes IT98K-499-39, Suvita 2, IT93K-503-1, and Mouride were found to be the most resistant genotypes to the development of the disease, with mortality below 10%.
The identification of sources of resistance to charcoal rot will enable the development of cultivars that are resistant to M. phaseolina, contributing to the sustainability of cowpea production chain and providing measures for producers to control this disease with reduced cost. The objective of this study was to identify lines that are resistant to charcoal rot, and the aim was to select parents for breeding programs for genetic resistance.

MATERIALS AND METHODS
The experiments were carried out from July 2018 to August 2019 in the Plant Pathology Laboratory and the greenhouse of Universidade Federal do Vale do São Francisco (UNIVASF) in Petrolina, Pernambuco, Brazil. The experimental design was a completely randomized design (CRD). One hundred lines of cowpea developed by Embrapa Meio-Norte (Teresina, Piauí, Brazil) were used ( Table 1). The experiment was divided into stages, each stage containing 20 lines per experiment. Each plot was a pot with two plants, with five replications (pot).For each line, there was a control (toothpick inoculated without the fungus). Isolates 59 of M. phaseolina were obtained from an infested area from the field of Agricultural Sciences Campus -Univasf and Isolate CMM 2106 was obtained from the Phytopathogenic Fungal Culture Collection of Professor Maria Menezes from Universidade Federal Rural de Pernambuco. These isolates were selected because they were more aggressive in previous studies (SOUZA et al., 2022).

Seeding of cowpea lines
Two seeds of each cowpea line were sown in plastic pots (1 L) containing substrate and soil mixture (1:1) previously autoclaved at 120 °C for 1 h twice (2 days); the sowing was done in greenhouse underscreens (Sombrite ® ), which retained 50% brightness. After sowing, the pots were irrigated twice daily until inoculation. After inoculation, the pots were irrigated once a day with a fixed volume of 180 ml of water.

Inoculum production
The infested toothpick method was used to inoculate M. phaseolina isolates (COHEN; ELKABETZ; EDELSTEIN, 2016). Isolates 59 and CMM 2106 were plated in Petri dishes containing potato dextrose agar (PDA) medium and maintained in a biological oxygen demand (BOD) at 25 °C for seven days for mycelium growth. Afterward, isolates were plated in new Petri dishes containing PDA medium and toothpick previously autoclaved. Petri dishes with a toothpick and the isolates were maintained in a BOD for 15 days until the fungus colonized all the surfaces of the plate and toothpick.

Inoculation using infested toothpick method
The inoculated toothpicks were used for plant inoculation 15 days after sowing by stabbing the crown approximately three cm above the ground. Plant height (cm) was evaluated after inoculation. Three evaluations were made every three days by measuring the lesion length (cm) caused by the fungus and plant height (cm) with a ruler. The variables obtained were lesion length, plant height, area under the disease progress curve (AUDPC), severity, and relative growth compared to the control (RGCC). The length of the lesion was converted to a note as described by You, Colmer and Barbetti (2011) where the plants were assessed for incidence/ severity of disease using 0 to 5 scale for lesions and discoloration on the hypocotyls; where: 0 = no disease; 1 = 1 cm lesion/discoloration; 2 = >1 to 1.5 cm; 3 = >1.5 -3 cm; 4 = >3 to 5 cm lesion/ discoloration or plant collapsed. Control is the treatment (accession) inoculated without the isolates of M. phaseolina.

Statistical analysis
Data from each experiment using 20 lines were subjected to analysis of variance, and the means were grouped using the Scott-Knott test (α=0.05) of the SISVAR software (FERREIRA, 2011). Assumptions underlying the analysis of variance has been checked.

RESULTS AND DISCUSSION
There was a significant difference (P<0.05) between lines 1 to 20 for the variable HD (Table 2) when inoculated with isolate 59 and CMM 2106, with averages ranging from 1.97 cm to 7.80 cm and 3.14 cm to 10.65 cm, respectively. Lines 61 to 80 also had a significant effect (P<0.05) on HD when inoculated with isolate 59, with averages ranging from 6.55 cm to 8.75 cm. However, HD was not different for lines 61 to 80 when inoculated with isolate CMM2106, with averages ranging from 4.65 cm to 10.50 cm. Among the lines that had a significant effect on HD when inoculated with isolate 59, the ones with the highest means were L1, L6, L7, L8, L9, L10, L11, L12, L20, L61, L64, L65, L71, L72, L73, L74, L75, L76, L77, L78, L79, and L80. For isolate CMM 2106, only lines L3, L10, L18, L25, L27, L29, L30, L31, L32, and L40 had the highest means. It has been reported in literature that plants, which are infected during their early stages of development, are smaller than normal in soybean crop (ISHIKAWA et al., 2018). Plant growth can be affected by the pathogen because M. phaseolina enters the host tissue by dissolving the cell wall through the secretion of toxins or enzymes, making plant development difficult, which consequently results in small plant height (MEDEIROS et al., 2015).
No difference was observed between lines for AUDPC (P>0.05). Severity for both isolates was only different in the experiment of lines 1 to 20. For isolate 59, severity ranged from 3.04% to 12.60% with line 11 having the highest mean severity (12.60%), whereas for isolate CMM 2106, the mean severity ranged from 1.79% to 14.31% with lines L1, L5, L7, L10, L11, L12, L14, L15, L16, L17, L19, and L20 having the highest averages ranging from 6.44% to 14.31%. Thus, line 11 had one of the highest averages of severity, and it can be characterized as a susceptible line for both isolates 59 and CMM 2106. The absence of immunity to this pathogen is common among legumes, as it is a generalist species (GARCÍA et al., 2019).
For length of lesion, no significant difference was observed between lines for most experiments (P >0.05). The average length of lesion for isolate 59 ranged from 0.86 to 1.88 cm for lines 1 to 20, 0.70 to 1.66 cm for lines 21 to 40, 0.52 to 0.91 cm for lines 41 to 60, and 0.71 to 1.46 for lines 61 to 80, and 0.48 to 1.02 cm for lines 81 to 100. For isolate CMM 2106, average lesion ranged from 1.20 to 4.0 cm for lines 1 to 20, 1.0 to 1.80 cm for lines 21 to 40, 0.40 to 2.80 cm for lines 41 to 60, and 1.20 to 2.80 cm for lines 81 to 100; for lines 61 to 80, only the score of the lesion was made. The hot and dry weather conditions (temperatures between 28 °C and 35 °C) are favourable to the development of M. phaseolina (CRUCIOL; COSTA, 2018). Recently, a study carried out with melon accessions showed that increase in temperature increased the severity of symptoms caused by M. phaseolina in most genotypes evaluated, thus the effect of the temperature on disease severity should be considered during the genetic studies (LINHARES et al., 2020). According to the UNIVASF Meteorology laboratory, the temperatures during the evaluation periods of the experiments were within ideal conditions for the development of the pathogen, with a maximum average temperature of 32.2 °C and a minimum of 21.1 ºC.
Analysis of lesions was performed by converting the lesion length into notes according to the scale of You, Colmer and Barbetti (2011). There were significant differences between lesion scores in lines 1 to 20 and 81 to 100. For lines 1 to 20, there was variability for both isolates (Table 3).
The highest average lesion scores were observed in CMM 2106 isolate, and it was considered the most aggressive isolate. Isolate CMM 2106 was obtained from the cowpea host plant, while isolate 59 was obtained from the common bean host plant (SOUZA et al., 2022). For isolates 59 and CMM 2106, 9 and 8 lines were obtained, respectively, with the lowest average scores of the injury caused by M. phaseolina (Table 3). Line 8 was the only one to be in the group with the lowest average scores for both isolates; therefore, it can be a promising source of resistance. The average scores for lines 81 to 100, inoculated with isolate 59, ranged from 1.00 to 1.75. Lines L81, L83, L84, L86, L87, L88, L89, L90, L91, L92, L93, L94, L95, L96, L97, L98, and L99 had the lowest averages. There was no variation for CMM 2106 in this step. For the lines evaluated during the other stages, there was no significant difference for both isolates (P> 0.05).
There was a significant difference in relative growth compared to control (RGCC) for both isolates. The lines considered most resistant were those which growth was not affected by inoculation. For lines 1 to 20, seven lines were considered resistant because they had an RGCC greater than 100% for isolate 59 (Figure 1 A). For isolate CMM 2106, 2 lines were considered resistant, which RGCC was higher by 206.19% compared to control ( Figure 1B). Of the lines 21 to 40 evaluated for isolate 59, 7 resistant lines were considered to have RGCC greater than 101% ( Figure 1C). For CMM 2106 isolate, only one line was resistant ( Figure 1D). In lines 41-60, 3 resistant lines were observed for isolate 59 ( Figure 1E) and had RGCC greater than 184%. Regarding the CMM 2106 isolate, 4 resistant lines were considered ( Figure 1F).
Among lines 61 to 80, one resistant line was identified, with an RGCC greater than 150% for isolate 59 ( Figure 1G). For isolate CMM 2106, 4 resistant lines presented an RGCC of 265% greater than the control ( Figure 1H). For lines 81 to 100, only one resistant line was identified for both isolates 59 ( Figure 1I) and CMM 2106, with RGCC greater than 245% and 286.85%, respectively ( Figure 1J). Among these lines, two are commercial cultivars: BRS Tumucumaque and BRS Pajéu, and were susceptible to charcoal rot for both isolates.
There are still few reports of cowpea lines that are resistant to M. phaseolina. Some studies reported genotypes that are moderately resistant or highly resistant to this pathogen. Muchero et al. (2011)   Lines 8, 10, and 19 had the lowest average scores (Table 2) with the highest RGCC greater than 100% for isolate 59 ( Figure 1A). The other lines had the lowest average scores and had their growth affected by the pathogen, that is, their RGCC was less than 100%. However, lines 6, 7, 9, and 11 were ranked to have the highest average of lesions, and their growths were not affected. For the isolate CMM 2106, only line 3 (Table 2) has the lowest averages of lesion score and was in the group that did not have its growth affected by the pathogen, with an RGCC greater than 206% (Figure 1). Lines 2, 13, and 15 had lower average scores and their RGCC ranged from 128.6% to 206%. Even though line 4 belonged to the highest average score, its height was not affected by the pathogen.
For both isolates, line 8 obtained the lowest average lesion scores; the line also had an RGCC of 109.63% for isolate 59 and 110% for isolate CMM 2106. Line 11 obtained one of the highest averages of the lesion scores and had greater severity for both isolates. Line 10 had a greater difference in height for the two isolates; however, this line belongs to the group with the highest severity for isolate CMM 2106. Lines 1 to 20, which had the highest scores of injuries, also had the highest severity. Lines 17 and 15, which had greater severity, had the lowest lesion scores for isolate CMM 2106. For isolate 59, line 11, which had greater severity, also had higher average lesion scores. The growth of lines L25, L26, L27, L29, L34, L38, L39, L44, L48, L55, L79, and 95 were not affected by the isolate 59; their average scores ranged from 1.00 to 1.60 and were also considered resistant. For isolate CMM 2106, the average scores of lines L24, L51, L63, L71, L76, L80, and L93 ranged from 1.00 to 2.33.
In soybean, a study for the identification of candidate genes involved in charcoal rot resistance and unravels the complexity of this resistance (COSER et al., 2017). Additional research is needed to determine resistance mechanisms in cowpea. The genetic entries identified in this paper will be useful for improving charcoal rot resistance in cowpea crop.

CONCLUSIONS
Promising cowpea lines identified as resistant sources of resistance to M. phaseolina can be used by farmers directly as new cultivars or in future hybridizations of cowpea genetic breeding programs.
The variables lesion scores and relative growth compared to control were more promising in assessing resistance of the cowpea lines to isolates 59, and CMM 2106 of M. phaseolina using the toothpick inoculation method.
The M. phaseolina isolate CMM 2106 is more aggressive than isolate 59.