Rhizobium phaseoli tolerant to insecticide on the growth of Phaseolus vulgaris

Rhizobium phaseoli tolerante a un insecticida en el crecimiento de Phaseolus vulgaris

Santoyo-Pizano, Gustavo

Michoacan University of San Nicolás de Hidalgo, México

Hernández-Mendoza, José Luis

National Polytechnic Institute, México

Márquez-Benavides, Liliana

Michoacan University of San Nicolás de Hidalgo, México

De Luna-Esquivel, Gustavo

Chapingo Autonomous University, México

Sánchez-Yáñez, Juan Manuel syanez@umich.mx

Michoacan University of San Nicolás de Hidalgo, México

This work is licensed under Creative Commons Attribution-NonCommercial 4.0 International.

The symbiosis between legumes and Rhizobium is essential in agriculture due to the positive impact it has on the cultivation and consumption of Phaseolus vulgaris and other legumes¹. Inoculating P. vulgaris seed with Rhizobium phaseoli avoids nitrogen hyperfertilization to the soil, in addition to ensuring the healthy growth and profitable yield of P. vulgaris¹ whose grain is considered one of the main in Mexico, in principle due to its high consumption and production, hence the economic importance².

One of the problems to ensure a healthy growth of P. vulgaris, is the control of insects-pests that damage the root, thus avoiding the beneficial action of R. phaseoli³, first to exert the infectivity or formation of nodules and the effectiveness or the ability of R. phaseoli to adequately supply the nitrogen (N) demand for healthy growth. Consequently, pesticides are applied that generally inhibit or kill R. phaseoli, however, the information in this regard is contradictory, since it is reported that the growth of R. phaseoli is inhibited with minimal concentrations of dichloro diphenyl trichloroethane (DDT) and folidol, being lower than those recommended in the field in the pest control of roots of P. vulgaris. While other research reported that Rhizobium spp., can be tolerant to pesticides without losing the infective and effective capacity^4,5. Thus, it has been reported that nodulation in the root system of P. vulgaris by R. phaseoli is inhibited or reduced due to the toxicity of these pesticides^6,7, which decreases the effectiveness or positive effect for the healthy growth of P. vulgaris, basically because they inactivate nitrogenase to fix molecular nitrogen (N.) or FN.^8,9.In that sense the objectives of this research was the selection and analysis diazinon-tolerant R. phaseoli for the healthy growth of P. vulgaris.

Origin of the R. phaseoli isolated. R. phaseoli were isolated from the roots of Phaseolus var Bayo, in congo red yeast extract mannitol agar (CRYEMA) with the following composition (g/L): K₂HPO₄ 0.5, MgSO₄ 0.2, NaCl 0.1, mannitol 1.0, yeast extract 1.0, congo red 10.0 mL/L (from a 1:500 solution), agar 20.0 g, 1000.0 mL distilled water, pH 7.0 according to Vincent¹⁰, collected from the experimental agricultural field of the National Institute of Forestry, Agricultural and Livestock Research (INIFAP), Aldama, Tamaulipas, Mexico, were designated with the codes: ReD, ReDa and ReD1, they are part of the collection of the Environmental Microbiology laboratory of the Institute of Biological Chemical Research (IIQB) -Universidad Michoacana de San Nicolás de Hidalgo (UMSNH)^1,10.

Selection of R. phaseoli tolerant to diazinon. In this trial, pesticides frequently applied in Aldama Tamaulipas, Mexico, were tested, such as: diazinon (O, O-diethyl O-2-isopropyl-6-methylpyridin-4-yl phosphorothioate) - Servin (1-naphthyl N- methyl carbamate), c/pesticide was added to the CRYEMA in concentrations of: 250, 300, 500 and 100 ppm, in a nephelometric flask, 1.0 mL equivalent to 1 x 10⁶ CFU/mL of R. phaseoli was inoculated per 100 mL of yeast mannitol extract broth (YMEB) plus diazinon or Servin in concentrations of: 200, 300, 400 and 1000 ppm, these flasks were incubated for 24 to 36 h in shaking/200 rpm, for adaptation and selection of R. phaseoli to the increasing concentration of diazinon and Servin. The adaptation of R. phaseoli to the increase in the concentration of each pesticide was achieved by detecting the growth expected with that increase, compared to the normal growth of R. phaseoli in the nephelometric flask in YMEB without diazinon or Servin under the same conditions. incubation time, temperature and time⁴.

Test of infectivity and effectiveness of R. phaseoli resistant to diazinon in P. vulgaris. The diazinon-tolerant strains of R. phaseoli were selected (since Servin inhibited the growth of R. phaseoli from the first concentration so it was no longer used).

Figure 1
Leonard’ jar

Therefore, the seeds of P. vulgaris var Bayo (donated by the Secretary of Agriculture, Livestock, Fisheries, Rural Development and Food of the Mexican government) were inoculated with R. phaseoli resistant to diazinon in Leonard's jars (figure 1) there the soil was placed in the upper part of Leonard's Jar and the mineral solution or water in the lower part of the Jar, both parts were connected with a 20 cm long cotton strip, for movement of the liquid by capillary action⁵, The soil where P. vulgaris was sowing had the following physicochemical properties: it was a lateritic with pH 6.64, clay texture 40.56 %, sand 0.76 %, silt 37.8 %, field capacity 30.08 %, organic matter humidity 4.57 %, poor in total nitrogen 0.1 %, field capacity 30.08 %, and cation exchange capacity of 4.61 mg/100 g. This soil was sieved with a mesh of No. 20, it was solarized 48 h at 70 °C, to avoid pests and diseases, Leonard's Jars were placed in the greenhouse under the following average micro-climatic conditions were: the temperature of 23.2 °C, with a brightness of 450 µmol•m^-2•s^-1 and a relative humidity of 67 %. P. vulgare with R. phaseoli was fed with a mineral solution with the following chemical composition: 1 molar of K₂HPO₄, KH₂PO₄, CaCl₂, MgSO₄, traces of FeSO₄, 1.0 mL of minor elements solution in 1000 mL of distilled water adjusted to pH 7 which was pasteurized (90 °C/10 min). Later 45 days later, the effect of diazinon-tolerant R. phaseoli strains in P. vulgaris was evaluated by means of the following response variables, plant height (PH), leaf diameter (LD) (cm), as well as the dry weight of the foliage (DWF), number of effective nodules in the root its dry weight and the dry weight of the root¹⁰. Evidence that R. phaseoli degraded (coometabolism) diazinon in culture medium.

The diazinon-tolerant R. phaseoli strains were inoculated in a nephelometric flask, with congo red mannitol yeast extract broth (CRMYEB) with diazinon, the flasks were shaken at 300 rpm until the stationary phase, by means of a viable count that was reached at 30 h of incubation according to the growth curve of R. phaseoli, in comparison with the same R. phaseoli in CRMYEB without diazinon. This test showed the coometabolism of R. phaseoli to degrade diazinon in combination with mannitol used as a carbon source ^4,11-13.

Table 1 shows the effect of diazinon-tolerant R. phaseoli strains on the growth of P. vulgaris as a function of the number of effective nodules that determine a plant health condition with 19, 20, 39 generated respectively by the strains of R. phaseoli resistant to diazinon called ReD, ReDa and ReD1. Regarding the color of P. vulgaris leaves, they were indicative that the isolated infected the roots of P. vulgaris formed red nodules (effective), which indicates the presence of leghemoglobin associated with its capacity to fix N₂ and the color intense green on the leaves¹.

Table 1
Effect of diazinon-tolerant Rhizobium phaseoli on the growth of Phaseolus vulgaris var Bayo

¹n=20*equal letters without statistical differences according to Tukey (p<0.05)

In table 2, where according to the literature it was observed that the diazinon-tolerant R. phaseoli coding as ReD, ReDa and ReD1 had a positive effect on the LD, and PH of P. vulgaris with 19.1, 24.9, 18 cm, as well as in the DWF, these numerical values were statistically different in comparison with the wild R. phaseoli isolates unable to grow in the presence of diazinon. Like the diazinon-tolerant .. phaseoli isolates in the number of effective red nodules, the dry weight that included that of the complete root, not statistical difference compared to the analogous numerical values registered in P. vulgaris with wild R. phaseoli non-diazinon tolerant.

Table 2
Comparison of the infectivity and effectiveness of wild Rhizobium phaseoli and diazinon-tolerant Rhizobium phaseoli on the growth of Phaseolus vulgaris var Bayo

¹n=20, *Equal letters without statistical differences according to Tukey (p<0.05).

Table 3 shows that R. phaseoli strains grown in CRYEMA with diazinon generated a number of colony forming units (CFU) with a statistically different numerical value compared to wild R. phaseoli isolated (wild) in CRYEMA without diazinon, which indicates that the isolates subjected to the maximum concentration of diazinon were physiologically adap-ted to the insecticide, although there was a reduction in the UFC R. phaseoli, since it is a natural selection which caused the death of the cells of R. phaseoli that are susceptible to diazinon toxicity.

Table 3
Comparison of the stability of resistence of Rhizobium phaseoli to diazinon compared to wild Rhizobium phaseoli

¹* average of 4 repetitions in CFU = Colony Forming Units x102/mL *CRYEMA = congo red yeast extract mannitol agar.

*Equal letters without statistical differences according to Tukey (p<0.05).

* Equal letters without statistical differences according to Tukey p005

Table 4 shows the growth as CFU, registered in diazinon-tolerant R. phaseoli isolates, the literature indicates that it may be due to an action of coome-tabolism when they use mannitol for the degradation of diazinon, in this table it was evident that when R. phaseoli grew in mannitol and diazinon, a decrease in the number of colonies was registered as CFU, because although R. phaseoli degraded it, it was toxic for a part of the total population of R. phaseoli, compared to when it grew in the CRYEMA without diazinon, which represents the amount of the expected CFU growth, while by suppressing mannitol and diazinon, the absence of both caused a drastic decrease in the R. phaseoli population, because the other components of CRYEMA: like yeast extract and Congo red were insufficient for R. phaseoli to reproduce at the level registered when the culture medium contained mannitol as sole source of carbon and energy¹.

Table 4
Diazinon coometabolism by insecticide tolerant Rhizobium phaseoli isolates comparad to the growth without insecticideon the wild Rhizobium phaseoli

¹CFU = Colony Forming Units/mL; *Equal letters without statistical differences according to Tukey (p<0.05).

Table 1 shows that a positive response of P. vulgaris was observed based on the number of effective nodu-les, which supports that diazinon-tolerant R. phaseoli conserved plasmids that contain infectivity and effectiveness in the roots of P. vulgaris, for healthy growth, because there was insufficient mineral nitrogen in the soil for the growth of P. vulgaris^1,5,6,12.

Table 2 shows that the diazinon-tolerant R. phaseoli strains conserved the plasmids responsible for nodulation or infectivity, which were red in color, indicating the presence of leghemoglobin, a protein related to the ability to fix N. (effectiveness ), consequently P. vulgaris synthesized enough chlorophyll, for the intense green color of the leaves, a phenological characteristic of a healthy growth when P. vulgaris is symbiosis with strains of R. phaseoli that are infective and effective ^7,10,13.

Table 3 shows the evidence that the growth of R. phaseoli in yeast mannitol extract agar (YMEA) with diazinon based on CFU, was the result of a natural selection of the physiological adaptation to diazinon, without the risk of losing the capability to grow in the presence of diazinon, the numerical value of the CFU, was reduced in relation to the same variable of CFU, of the wild strains of R. phaseoli, because during the selection a part of this population susceptible to diazinon died, therefore, the number of CFU of R. phaseoli tolerant to diazinon was lower^14-16. As has been reported for other genera, species of the Rhizobiaceae family such as Bradyrhizobium spp., which also co-metabolize and/or mineralize other applied pesticides, in conventional agriculture in the control of insects-pests that attacking the roots of legumes^15,16. The capability to degrade insecticides similar to diazinon, is a common biochemical property in nature according to those reported for other bacterial groups other than R. phaseoli such as: Pseudomonas, as well as actinomycetes: Streptomyces, Micromonospora, Thermoactinomyces, Nocardia and Mycobacterium^4,12,13 and other genera and species of soil fungi, and/or those endophytic living in association with plant roots of the type: Aspergillus fumigatus, Cunninghamella elegans¹⁷, Penicillium citreonigrum¹⁸. Rhizopus nodosus, includes the human pathogenic enterobacteria: Escherichia coli, Salmonella bongori that are capable of mineralizing diazinon, the above supports this is because both eukaryotes and prokaryotes have this genetic property of resistance to diazinon, because they are related to human activities of plant and water management that are part of agriculture^19-21.

Table 4 shows the tolerance of R. phaseoli to diazinon was the results of the physiological adaptation capacity of this genus and species, which also has the codified genome using as only source carbon some compounds similar to diazinon, so if there is another simple carbon source that, when is oxidized, made easy the degradation of the pesticide, a biochemical action known as coometabolism. What was showed that when R. phaseoli tolerant to diazinon grew in CRYEMA and diazinon, compared to the inhibition of growth of R. phaseoli in CRYEMA without mannitol or diazinon^4,8,11,14. The selection of strains of R. phaseoli tolerant to pesticides is useful in conventional agriculture, its has been reported on Bradyrhizobium japonicum strains for producing Glycine max to avoid attacking of pest insects ^15,22,23. For this reason R. phaseoli is recommended in agricultural issues where P. vulgaris has a problem with insect pest destroying roots and limiting its healthy growth, underline due diazinon is applied for producing P. vulgaris in doses similar to those evaluated in this research¹², since diazinon is applied at the begining growth steps of P. vulgaris without evidence that its persistence in P. vulgaris tissues and/or seeds¹⁴. Based on the above, its concluded that it is possible to isolate diazinon-tolerant strains of R. phaseoli that are highly infective and effective for a healthy growth of P. vulgaris without risk of lost caused by pest insects destroying its root system.

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