Root-knot nematodes, Meloidogyne spp., constitute one of the four primary genera of economically significant plant-parasitic nematodes in Brazil, particularly affecting key crops in the country. Susceptible soybean cultivars experience productivity losses ranging from 30% to 90% due to nematode attacks, with M. javanica identified as the most detrimental species, causing average losses of 40% in sandy or medium sandy soils (Rocha and Dias-Arieira 2023). Recent surveys indicated that Meloidogyne species are present in almost 30% of soybean samples collected throughout Brazil in 2022. However, when focusing on the southern region alone, the frequency of occurrence exceeds 60% (Rocha and Dias-Arieira 2023).
Efforts to reduce phytonematode populations involve the integration of various control methods, encompassing cultural, genetic, chemical, and biological management (Ferraz et al. 2010). Nasu et al. (2019) emphasize that successful management depends on the compatibility of these methods, with the adverse effects of one on the other being undesirable.
Biological control of nematodes has witnessed substantial growth in Brazil, with fungi such as Purpureocillium lilacinum, Trichoderma spp. and Pochonia chlamydosporia, as well as bacteria from the genera Bacillus spp. and Pasteuria spp. emerging as prominent biological control agents (Dias-Arieira et al. 2023; Machado 2022; Nasu et al. 2019). However, the efficacy of biological nematicides is influenced by both biotic factors, like the soil microbial community, and abiotic factors such as temperature, humidity, pH, environmental residues, and compatibility with chemical products used for crop management, including herbicides (Reis et al. 2013).
The application of herbicides for weed control can impede the development or action of microorganisms utilized in the biological management of nematodes, diminishing their effectiveness. This has been observed with Trichoderma spp. by Reis et al. (2013) and with Bacillus sp. by Gonçalves et al. (2020). Therefore, it is imperative to study the compatibility of biological products with agrochemicals to ensure the viability and practicability of using microorganisms on a large scale, contributing to the development of effective and integrated management strategies (Nasu et al. 2019).
Some herbicides, notably glyphosate, have reported adverse effects on various nematode species. Glyphosate has been shown to interfere with the growth, fecundity, and reproduction of Caenorhabidits elegans by Kronberg et al. (1994). Subsequent studies revealed a high production of reactive oxygen species in C. elegans treated with glyphosate, similar to observations with paraquat-treated nematodes (Kronberg et al. 2018; Park et al. 2009). Exposure of C. elegans to glyphosate inhibits mitochondrial respiration and leads to the accumulation of hydrogen peroxide, as demonstrated by Bailey et al. (2018). Additionally, several authors have reported the production of reactive oxygen species in nematodes exposed to insecticides such as chlorpyrifos and monocrotophos (Leelaja and Rajini 2013).
The objective of this study was to assess the in vitro impact of herbicides commonly used in soybean cultivation on the development of Bacillus firmus, Purpureocillium lilacinum, Pochonia chlamydosporia and Trichoderma harzianum, and their influence on Meloidogyne javanica.
For in vitro compatibility tests, B. firmus strain I-1582, and the fungi P. chlamydosporia strain Pc 10, T. harzianum isolate IBLF 006, and P. lilacinum strain Pae 10, isolated from the commercial products Votivo Prime® (BASF), Rizotec® (Corteva), Ecotrich®, and Nemat® (Ballagro), respectively, were used. The experimental design followed a completely randomized design with nine treatments and five replicates, considering each Petri dish (90 mm in diameter) as an experimental unit. These experiments were repeated twice.
The treatments included a control group (without herbicide addition) and herbicide applications at specific doses: diclosulam (Spider®, Corteva) at 26.88 g a.i. ha-1, sulfentrazone (Boral®, FMC) at 300 g a.i. ha-1; flumioxazin (Sumyzin®, Sumitomo) at 50 g a.i. ha-1; imazethapyr (Vezir®, Adama) at 106 g a.i. ha-1; S-metolachlor (Dual Gold®, Syngenta) at 1440 g a.i. ha-1; pyroxasulfone (Yamato®, Ihara) at 150 g a.i. ha-1; imazethapyr + flumioxazine (Zethamaxx®, Sumitomo) at 127.2 + 60 g a.i. ha-1, and glyphosate (Xeque-Mate®, Ihara) at 1240 g a.i. ha-1. The herbicides were diluted in culture medium post-sterilization in an autoclave but before solidification to maintain the final concentration without active ingredient degradation.
For B. firmus, 100 µL of bacterial suspension with a concentration of 105 CFU mL-1 was transferred to Petri dishes containing the T.S.A. culture medium, and after 72 hours of incubation at 28 ± 2 ºC in a B.O.D., colonies were counted, and values were transformed into a logarithmic ratio (LogCFU mL-1). P. lilacinum, P. chlamydosporia, and T. harzianum were evaluated by placing 7 mm diameter discs containing the P.D.A. culture medium with mycelium in the center of Petri dishes. Incubation occurred at 28 ± 2ºC with a 12-hour photoperiod. Daily measurements of fungal mycelial growth in both perpendicular directions of the Petri dish were taken to calculate the Mycelial Growth Speed Index (IVCM=∑( D-Da)/N, where D is the current diameter of the colony, Da is the diameter of the colony on the previous day and N is the number of days after inoculation) (Olher et al. 2021) and Inhibition of Mycelial Growth (ICM). For this, the values of colony diameters from the last day of incubation were used, according to the formula described by Mohiddin and Khan (2013): I=(C-T)/C x100, where I corresponds to the percentage of inhibition, C to the diameter of the mycelial growth of the control and T to the diameter of the mycelial growth of the herbicide treatment.
To assess the impact of herbicides on M. javanica, the nematodes necessary for the study were extracted from soybean roots using the optimized Baermann funnel methodology (Machado and Silva 2019), ensuring the extraction of active nematodes. Nematodes were quantified in a Peters chamber under a light microscope, and the suspensions were calibrated to 50 nematodes per ml. The nematodes were then distributed in test tubes with a final suspension volume of 6 mL. Initially, 1 mL containing 50 nematodes was pipetted into each replicate, followed by the addition of 5 mL of the herbicide suspension at the doses previously described, considering a spray volume of 200 L ha-1. The test tubes were maintained in a B.O.D at a temperature of ± 25° C. A control treatment, containing only the nematode suspension and water, was included. Each tube represented an experimental unit, with five replicates. This experiment was repeated twice.
After 24 and 48 hours of exposure, nematodes were counted under an optical microscope, categorizing those showing no movement as dead and those maintaining normal behavior, characteristic of the species, as alive. Mortality rates were expressed as a percentage for each treatment and evaluation date.
The data were submitted to a Shapiro Wilk test at a significance level of 5% to verify the normality of the residuals. As the data did not meet normality, the Kruskal-Wallis non-parametric test at a significance level of 5% was employed. All the analyses were performed using the statistical software R (R Core Team 2020) using the packages MASS (Venables and Ripley 2002) and ExpDes (Ferreira et al. 2013).
The development of B. firmus was negatively influenced by the pre-emergent herbicides imazethapyr, S-metolachlor, and glyphosate, which completely inhibited bacterial growth in both experiments (Tables 1 and 2). Treatments with pre-emergent herbicides diclosulam, sulfentrazone, flumioxazine, piroxasulfone, and the mixture of imazetapyr with flumioxazine did not interfere with bacterial multiplication. Similar adverse effects were also observed by Dennis et al. (2018) when studying the impact of glyphosate, glufosinate, paraquat, and diquat on the diversity and functionality of bacteria and nematodes in soil from a banana plantation. Gonçalves et al. (2020) also reported a negative effect of glyphosate on the growth of a Bacillus isolate.
In the case of P. chlamydosporia (Tables 1 and 2), all the treatments, except diclosulam, exhibited lower IVCM values, compared to control. Interestingly, diclosulam did not interfere with the growth of this fungus, showing an IVCM of 4.08 and 3.98 mm day-1, respectively for experiments 1 and 2, similar to the control without herbicides (4.12 and 4.06 mm day-1). The ICM index showed the opposite classification of treatments, in which S-metolachlor showed the highest percentage of inhibition of P. chlamydosporia in both experiments. Carvalho et al. (2022) observed growth inhibition of P. chlamydosporia with the application of various fungicides, insecticides, and the nematicide oxamyl.
For P. lilacinum (Tables 1 and 2) S-metolachlor completed halted its development in both experiments. All the treatments reduced the IVCM of P. lilacinum in experiment 2, but diclosulam did not have this effect in experiment 1. As observed for P. chlamydosporia, also for P. lilacinum the treatments had an opposite effect on ICM compared to IVCM. Kerry et al. (2009) reported the sensitivity of P. chlamydosporia and P. lilacinum to various concentrations of fungicides and herbicides like bentazone, pendimethalin, and metribuzin. The impact of glyphosate on the fungi P. clamydosporia and P. lilacinum aligns with the findings of Mensin et al. (2013), who observed reduced fungal growth and alterations in mycelium morphology.
Concerning T. harzianum (Tables 1 and 2), S-metolachlor prevented mycelium growth in both experiments, while diclosulam had no impact on growth rate in experiment 1. All other treatments reduced the IVCM of T. harzianum in both experiments. Gayatri et al. (2016) assessed the in vitro compatibility of fungicides and insecticides with fungi, including T. harzianum, T. viride, P. lilacinus, P. chlamydosporia, and Bacillus species, reporting high toxicity of certain products on the tested microorganisms. However, a study by Bharadwaz et al. (2023) found that the herbicide glyphosate was compatible with T. viride, highlighting the variability in compatibility among Trichoderma species.
In examining the impact of herbicides on M. javanica (Table 3), imazethapyr proved to be lethal within the first 24 hours, causing 100.00% and 99.72% mortality among juveniles, respectively in experiments 1 and 2. S-metolachlor was responsible for 31.49% and 29.49% mortality in the initial assessment, respectively. Conversely, herbicides such as diclosulam, sulfentrazone, flumioxazine, piroxasulfone, imazethapyr + flumioxazine, and glyphosate did not differ significantly from each other or the control. In the subsequent evaluation after 48 hours of exposure, imazethapyr maintained the mortality rate of M. javanica at 100.00% in experiment 1 and increased to 99.72% in experiment 2. Flumioxazine exhibited the second highest mortality rate (90.21% and 61.04%, respectively in experiments 1 and 2) at 48 hours. At 48 hours, all of the herbicides increased the mortality rates compared to the control, except diclosulam in experiment 2.
Past research suggests that the impact of herbicides on nematodes depends not only on the active ingredient but also on the specific nematode species targeted (Weischer and Müller 1985). Mesnage et al. (2015), in a review on the effect of glyphosate on non-target organisms, noted that glyphosate salts exhibit low or very low toxicity in animals. However, in commercial formulations with the added surfactants and adjuvants, the penetration of the active ingredient through the cell membrane is facilitated, resulting in increased mortality (Haefs et al. 2002; Marc et al. 2002). In a similar vein, Yeh et al. (2018) found that the herbicide pendimethalin caused 100% mortality of M. incognita and P. coffeae, while glyphosate caused mortality only in P. coffeae, with no significant effect on M. incognita. Jordaan and de Waele (1988) observed no adverse effects on the penetration and reproduction of Pratylenchus zeae in maize with the application of atrazine, alachlor, EPTC and 2,4-D, in contrast to Ozman and Zohdy (1982), who reported that trifluralin and paraquat reduced M. javanica infection in tomato plants by 38% and 15%, respectively.
Table 1. Colony-forming units (CFU log mL-1) of Bacillus firmus and Mycelial Growth Speed Index (IVCM, in mm day-1), and percentage of Growth Inhibition (ICM, in %) of Pochonia chlamydosporia, Purpureocillium lilacinum, and Trichoderma harzianum under different herbicide treatments, under in vitro conditions. Experiment 1.
|
Treatments
|
Dose
(g a.i. ha-1)
|
Bacillus firmus
|
Pochonia chlamydosporia
|
Purpureocillium lilacinum
|
Trichoderma harzianum
|
|
UFC
|
IVCM
|
ICM
|
IVCM
|
ICM
|
IVCM
|
ICM
|
|
Control
|
-
|
6.86 c
|
4.12 a
|
0.00 i
|
3.93 a
|
0.00 f
|
56.25 a
|
0.00 g
|
|
Diclosulam
|
26.88
|
7.07 a
|
4.08 a
|
4.20 h
|
3.85 a
|
4.17 e
|
55.75 a
|
2.96 f
|
|
Sulfentrazone
|
300
|
6.87 bc
|
0.78 g
|
81.63 b
|
2.05 d
|
49.40 b
|
13.50 f
|
71.11 b
|
|
Flumioxazina
|
50
|
6.86 c
|
3.28 c
|
20.47 f
|
3.17 b
|
22.02 d
|
44.08 b
|
22.96 e
|
|
Imazetapyr
|
106
|
0.00 d
|
1.50 f
|
65.43 c
|
2.60 c
|
33.93 c
|
26.75 d
|
51.36 c
|
|
S-metolachlor
|
1440
|
0.00 d
|
0.00 h
|
100.00 a
|
0.00 e
|
100.00 a
|
0.00 g
|
100.00 a
|
|
Piroxasulfone
|
150
|
6.94 b
|
3.68 b
|
12.67 g
|
3.24 b
|
19.05 d
|
30.33 c
|
41.97 d
|
|
Imazetapyr + Flumioxazina
|
127.2 + 60
|
7.00 a
|
3.00 d
|
30.07 e
|
2.46 c
|
36.31 c
|
31.58 c
|
44.44 d
|
|
Glyphosate
|
1240
|
0.00 d
|
2.50 e
|
30.07 d
|
2.60 c
|
35.12 c
|
18.42 e
|
67.16 b
|
Each value represents the mean of 5 replicates. Means followed by the same letter do not differ from each other according to the non-parametric test of Kruskal-Wallis at a 5% significance level.
Table 2. Colony-forming units (CFU log mL-1) of Bacillus firmus and Mycelial Growth Speed Index (IVCM, in mm day-1), and percentage of Growth Inhibition (ICM, in %) of Pochonia chlamydosporia, Purpureocillium lilacinum, and Trichoderma harzianum under different herbicide treatments, under in vitro conditions. Experiment 2.
|
Treatments
|
Dose
(g a.i. ha-1)
|
Bacillus firmus
|
Pochonia chlamydosporia
|
Purpureocillium lilacinum
|
Trichoderma harzianum
|
|
UFC
|
IVCM
|
ICM
|
IVCM
|
ICM
|
IVCM
|
ICM
|
|
Control
|
-
|
6.82 b
|
4.06 a
|
0.00 i
|
4.08 a
|
0.00 h
|
54.33 a
|
0.00 i
|
|
Diclosulam
|
26.88
|
6.95 a
|
3.98 a
|
2.20 h
|
3.88 b
|
2.91 g
|
52.46 b
|
4.18 h
|
|
Sulfentrazone
|
300
|
6.91 a
|
0.64 g
|
84.00 b
|
2,12 g
|
47,02 b
|
14.24 f
|
69.93 c
|
|
Flumioxazina
|
50
|
6.91 a
|
3.28 c
|
20.20 f
|
3.28 c
|
17.73 f
|
43.80 c
|
23.28 g
|
|
Imazetapyr
|
106
|
0.00 c
|
1.48 f
|
63.50 c
|
2.50 e
|
37.72 d
|
28.13 e
|
49.89 d
|
|
S-metolachlor
|
1440
|
0.00 c
|
0.00 h
|
100.00 a
|
0.00 h
|
100.00 a
|
0.00 g
|
100.00 a
|
|
Piroxasulfone
|
150
|
6.92 a
|
3.50 b
|
13.03 g
|
3.25 c
|
18.80 f
|
31.96 d
|
39.98 f
|
|
Imazetapyr + Flumioxazina
|
127.2 + 60
|
6.94 a
|
2.88 d
|
27.10 e
|
2.25 f
|
44.14 c
|
32.15 d
|
43.81 e
|
|
Glyphosate
|
1240
|
0.00 c
|
2.42 e
|
39.86 d
|
2.60 d
|
34.86 e
|
13.81 f
|
75.24 b
|
Each value represents the mean of 5 replicates. Means followed by the same letter do not differ from each other according to the non-parametric test of Kruskal-Wallis at a 5% significance level.
Table 3. Percentage of mortality of second-stage juveniles of Meloidogyne javanica under the action of herbicides after 24 and 48 hours, under in vitro conditions.
|
Treatments
|
Dose (g i.a. ha-1)
|
Experiment 1
|
Experiment 2
|
|
24 hours
|
48 hours
|
24 hours
|
48 hours
|
|
Control
|
-
|
0.00 c
|
2.26 e
|
0.00 c
|
1.42 f
|
|
Diclosulam
|
26.88
|
0.92 bc
|
5.55 d
|
0.82 bc
|
3.81 ef
|
|
Sulfentrazone
|
300
|
0.75 bc
|
6.34 d
|
0.69 bc
|
4.41 de
|
|
Flumioxazina
|
50
|
0.82 bc
|
90.21 ab
|
0.85 bc
|
61.04 c
|
|
Imazetapyr
|
106
|
100.00 a
|
100.00 a
|
99.72 a
|
99.91 a
|
|
S-metolachlor
|
1440
|
31.49 a
|
57.07 b
|
29.49 a
|
50.13 b
|
|
Piroxasulfone
|
150
|
0.69 bc
|
11.33 c
|
1.16 bc
|
9.24 cd
|
|
Imazetapyr + Flumioxazina
|
127.2 + 60
|
1.30 b
|
6.16 d
|
1.14 bc
|
5.78 de
|
|
Glyphosate
|
1240
|
1.02 bc
|
9.60 c
|
1.41 bc
|
7.38 de
|
Each value represents the mean of 5 replicates. Means followed by the same letter do not differ from each other according to the non-parametric test of Kruskal-Wallis test at a 5% significance level.
In a meta-analysis examining the impact of various herbicides on nematode communities in soils, Zhao et al. (2013) discovered that herbicides have a negative influence on populations of predatory nematodes. This effect is likely attributed to disruptions in soil food chains. Additionally, the study noted an increase in populations of bacteriophagous nematodes, while populations of fungivores tended to decrease. The research found no significant effects on populations of omnivorous nematodes and phytoparasites.
Kornobis (2000) proposed that the impact of herbicides on nematodes primarily occurs during the early stages of their development, influencing them throughout the plant. This notion is indirectly supported by studies demonstrating the effect of herbicides on juvenile hatching, particularly for nematode species that lay eggs within root tissues (Weischer and Müller 1985; Wong et al. 1993; Beane and Perry 1990; Payan et al. 1987). Dmowska and Koznowska (1983) also suggested that soil may act as a buffer against the effect of herbicides on nematodes.
Overall, diclosulam exhibited the greatest compatibility with all bionematicides, while S-metolachlor completely inhibited the growth of the biological control agents tested and lead to high mortality of juveniles of M. javanica. Imazethapyr and flumioxazine were also identified as harmful to the nematode.
It is concluded that agrochemicals, including herbicides, can impact the growth and development of biocontrol agents, causing abnormalities in non-target organisms and reducing ecosystem biodiversity (Dennis et al. 2018; Zhao et al. 2013). While in vitro evaluations provide an initial understanding of herbicide influence on microorganisms used in biological control of nematodes and on nematodes themselves, further greenhouse and field studies tailored to Brazilian conditions are crucial. These studies will help comprehend the impacts of herbicides on biocontrol agents and M. javanica, considering potential variations in active ingredient concentrations at the soil-plant interface.