Antifungal Effects of Paraquat and Glyphosate on Rhizoctonia solani (Kühn) in Potato in vitro Condition

Potato is one of the main crops worldwide. It this research, antifungal activity in vitro of paraquat and glyphosate were evaluated for Rhizoctonia solani control. R. solani was identified from potato tubers collected out from at open markets in Saltillo, Coahuila, Mexico. Two types of herbicides were applied: paraquat and glyphosate, at four different dosage treatments of: 10, 100, 1 000 and 10 000 μL. One 5 mm diameter PDA disc with R. solani mycelium was placed at the center of the Petri dish, with a radial registry fungal every 24 h for 192 h. Pathogen was identified by morphological criteria and the data was evaluated randomly with a factorial arrangement, on which, herbicides represented factor A and dosage treatments were represented by factor B. Thus experimental design had two levels for factor A and five levels for factor B with six replications. The results were analyzed by the SAS version 9.1 statistical program, the mean separation with the Tukey test (p=0.05). Glyphosate achieved inhibition of R. solani by 35.5882% and paraquat up to 80.0399%. Results reveal the importance of the need for more studies of these herbicides as fungicides. High concentrations of paraquat (10 000 μL) inhibits R. solani, and glyphosate does not affect R. solani mycelium development at low dosages (10 and 100 μL) and inhibits it at higher doses (10 000 μL).


Introduction
Potato (Solanum tuberosum L.) is one of the four crops of greater importance in the world, just after rice (Oriza sativa L.), wheat (Triticum aestivum L.) and corn (Zea mays L.), with a production of 368 168 914 t in 2018 and in Mexico was 1 802 592 t (FAOSTAT, 2020), however, potato will be one of the key crops in reducing hunger and food security across the globe (Hussain et al., 2018). R. solani cause widespread soil-borne diseases is responsible for causing significant economic losses in many important field and horticultural crops all over the world (Grosch et al., 2004;Elsharkawy et al., 2014). Furthermore, is responsible for potato losses of 70% (Carling et al., 1989;Wilson et al., 2007), if you take that 70% loss, during that cicle, 1 261 814.4 t was lost in Mexico and 257 718 239.8 t worldwide.
Pesticides are chemical products used in agriculture to protect crops from pests (WHO, 2020). Glyphosate [N-(phosphonomethyl) glycine] is a broad-spectrum herbicide and the more is one of the more frequently used herbicides worldwide (Vila-Aiub et al., 2008;Duke & Powles, 2008;Duke, 2018), in forestation, aquatic weed control and agriculture (Bórtoli et al., 2012), since its introduction in 1974 by the Monsanto company (now Bayer) with the commercial product Roundup (Woodburn, 2000). This herbicide disables, the enzyme 5-enolpyruvylsiquimato-3-synthetize phosphate, which prevents the union of metabolite phosphoenolpyruvate in the active site of the enzyme (Carrera & Carreras, 2011;Salazar & Aldana, 2011). Paraquat [Dichloride of N, N'-dimethyl-4, 4'-bipyridylium] is a non-selective contact herbicide, in 1969 was introduced to Mexico and to this day, despite its high toxicity, it is one of the more used compounds for the control of broadleaf and pasture weeds (Herná ndez & Martinez, 2006). groups (AG 1-13 and BI), which have been divided based on hyphal anastomosis behavior, cultural morphology, host range, pathogenicity and other characters (Ogoshi, 1976;Carling et al., 1994Carling et al., , 2002. R. solani is one of the most important soil pathogen fungi of the world, in cultivated and not cultivated soil, causing diseases in several crops as potato, beans, tomato, among others (Montealegre et al., 2003;Meza-Moller et al., 2007). Studies have shown effects of herbicides on plant pathogens in vitro, as the inhibition of growth and reproduction of the pathogen (Katan & Eshel, 1973). Due the above mentioned, the objective of this research was to evaluate the antifungal activity of glyphosate and paraquat in vitro for R. solani control.

Pathogen Isolation and Identification
Potato tubers with R. solani (sclerotia) symptoms were collected from open markets in Saltillo, Coahuila, Mexico, 1 cm cuts with healthy and sclerotia and tissue, disinfected with 2% sodium hypochlorite for 5 min, and washed two times with sterile distilled water for 5 min. Four cuts equidistant manner in were placed in Petri dishes with PDA kept at 28 ± 2 °C. R. solani identification was determined following Sneh et al. (1991) keys. Purification was done through a spearhead and isolations were kept at 6 ± 2 °C.

PDA preparation with several herbicide dose levels
Several herbicide dosage levels in PDA culture medium (200 and 300 mL of PDA in glyphosate and paraquat respectively), were added once the sterilized environment was set at 38 ± 2 °C, poured into Petri dish in a 20 mL average per plaque. Dhingra & Sinclair (1985) food poisoning technique was used placing 5 mm diameter PDA discs with R. solani mycelium, at the center of the Petri dish with contaminated PDA, kept at a 26 ± 2 °C for eight days.

Evaluation
Mycelial growth was measured daily with the support of a vernier pointing to the four cardinal points (N, S, E, W), during 192 h. Data collected was evaluated randomly in a factorial arrangement, factor A for herbicides and factor B the treatments, with a two level in factor A and five levels for factor B, with six replications per treatment. Results were analyzed through the statistical computer program SAS version 9.1 (SAS Institute, 2002), and mean separation using the Tukey test (p=0.05).

Results and Discussion
The purified colonies showed a dark brown mycelial growth, this pathogen displayed a mature mycelium forming 90° angle, branches, moderately wide and sclerotia hyphaes, characteristic of R. solani, results that agree with to Nicoletti et al., (1999) Misawa et al., (2018). According to statistical analysis (p<0.0001), with the resulting coefficient of variation of 3.5053, there is a statistical difference between herbicides used; glyphosate with a 2.73.75 radial growth and paraquat with 0.8483 (Tab. 2), the Petri dish radio was 4.25 cm (100%), in general the glyphosate achieved R. solani inhibition by 35.5882% and paraquat up to 80.0399%.
In this research, higher dosages gave better results (10 000 μL), results obtained are opposed those to Vargas et al. (2002) who reported the lowest glyphosate dosage accomplishing inhibition of R. solani growth. (2.500 mg/L) and 300 mg/L (highest dosage) concentration, which had no effect on R. solani growth. Harikrishnan & Yang (2001) found no negative effect of glyphosate on vegetative growth of several R. solani isolates and anastomosis groups, however, inert ingredients in several formulations are suggested to be responsible of effect of glyphosate in the inhibiting mycelial growth and spore germination (Morjan et al., 2002).
Glyphosate does not affect R. solani mycelium development at low dosages (10 and 100 μL) and inhibits it at higher doses (10 000 μL).