The mealy mealybug
In cassava,
Even in other cultures, the management of these insects is considered unsatisfactory, due to their underground their habits, which impedes control by most natural enemies and agricultural pesticides (Alves and Moino, 2009; Guide et al., 2016; Souza and Ribeiro, 2003).
However, underground scale insects that attack roots can be easy targets for entomopathogenic nematodes (EPNs) (Rhabditida: Heterorhabditidae and Steinernematidae), which are organisms found in the soil and used for the control of insects that remain at least one phase of their life cycle in this environment, as is the case of mealybugs of the genus
Studies to assess the pathogenicity of EPNs on mealybugs of the genus
Thus, this study aimed to evaluate the virulence of nine isolates of the species
The insects (females of
The nematodes used in the tests were purchased from the research institutions EMBRAPA TRIGO (Passo Fundo, RS), the Federal University of Lavras (Lavras, MG), and the Instituto Biológico (Campinas, SP) (Table 1). In the laboratory, the isolates were multiplied according to the methodology described by Molina and López (2001), using last instar larvae of
Isolates, species, and collection sites of
Isolate | Species | Reference | Collection location |
---|---|---|---|
RSC05 |
|
Andaló et al. (2006) | Benjamin Constant – AM – Brazil |
GL (Alho) |
|
Andaló et al. (2009) | Lavras – MG – Brazil |
JPM4 |
|
Molina et al. (2005), Andaló et al. (2014) | Lavras – MG – Brazil |
RSC 03 |
|
Andaló et al. (2006) | Benjamin Constant – AM – Brazil |
JPM3 |
|
Molina et al. (2005), Andaló et al. (2014) | Lavras – MG – Brazil |
IBCB-n40 |
|
Dell’Acqua et al. (2013) | Taboporã – SP – Brazil |
IBCB-n46 |
|
Dell’Acqua et al. (2013) | Santo Antônio de Posse – SP– Brazil |
IBCB-n44 |
|
Dell’Acqua et al. (2013) | Santa Adélia – SP – Brazil |
NEPET11 |
|
Ribeiro (2018) | Palmeira das Missões – RS– Brazil |
IBCB-n05 |
|
Dell’Acqua et al. (2013) | Itapetininga – SP – Brazil |
EPN isolates (Table 1) were multiplied according to the methodology mentioned above and kept in aeration in 1-liter Erlenmeyer flasks, for a maximum of one week before their use in the assays. Each treatment (10 isolates + control) was repeated five times totaling 55 parcels, which consisted of a 100 mL plastic cup containing 100 g of sterile sand (autoclaved) and a piece of pumpkin, of 3 cm² paraffined on the opposite side of the bark to prevent fungal growth. The insects (10 females) were placed on the bark, and then covered with sand. The nematode isolates were inoculated with the aid of a micropipette in aqueous suspension at a concentration of 3.3 × 103 infective juveniles (IJs)/cup (equivalent to 100 IJs/cm² of cup surface), diluted in 10 mL of distilled water (10% of relative to the sand mass). In the control treatment, the glasses received only distilled water.
The cups were closed with perforated plastic lids and kept in a climate chamber with temperature control (25 ± 1°C) and in the dark. The evaluation took place five days after inoculation, when dead insects were counted, and visual infection with nematodes was confirmed by dissecting the insects under a stereomicroscope.
The
Two tests were performed evaluating vertical and horizontal dispersal of the EPNs, using
To evaluate vertical dispersal, columns were assembled with 750-mL plastic cups, totaling 30 cm in height (3 cups). The substrate used was autoclaved fine sand, previously moistened with distilled water at 10% of the weight, which was placed in the three stacked cups and with their bottom parts removed to form an interconnected column. To assess the dispersal of the EPNs, four pieces of paraffined pumpkin were placed, which were arranged in four depths (0, 10, 20, and 30 cm, in ascending order from the top to the bottom of the column). Each piece of pumpkin received five
In the test to evaluate the horizontal dispersal, a PVC tube structure was used, which was cut to be 5 cm length with 14.5 cm in diameter. The substrate (paraffined pumpkin) containing the insects (10 females) was prepared in the same manner as in the previous test and arranged in four different positions correspond in the circle N, S, E, and W (about North, South, East, and West) arranged around the perimeter, at an angle of 90 degrees. The nematode isolate was applied, at the concentration 100 IJs/cm² (1.65 × 104 IJs/plot) in a suspension volume of 5 mL at the center.
In both dispersal tests, each treatment was repeated four times and the mortality of the mealybugs was evaluated five days after application. In both methodologies, the mortality was compared with a control treatment, which received only distilled water. The mortality of mealybugs was confirmed by dissection using a stereomicroscope.
For the compatibility test, phytosanitary products were registered and/or under study for registration in the cassava culture (Table 2). The doses were calculated according to the manufacturer’s recommendation, with a solution volume equal to 144 liters per hectare and the isolate used was the NEPET11 (
Trade name, biological activity, active ingredient, and dose of commercial product of phytosanitary products registered or in the process of registration for cassava culture, evaluated for compatibility with
Commercial name | Biological activity | Active ingredient | Situation | Dose/Haa | Dose/Lb |
---|---|---|---|---|---|
Curyom 550 EC | Insecticide | Lufenuron 50 g/L + profenofos 500 g/L | Registered | 300 mL | 2.08 mL |
Gaucho FS | Insecticide | Imidacloprid 600 g/L | Not registered | 360 mL | 2.50 mL |
Actara 250 WG | Insecticide | Thiamethoxam 250 g/kg | Not registered | 150 g | 1.04 g |
Tiger 100 EC | Insecticide | Pyriproxyfen 100 g/L | Not registered | 220 g | 2.50 g |
Nomolt 150 | Insecticide | Teflubenzuron 150 g/L | Registered | 300 mL | 1.50 mL |
Cipermetrina 250EC | Insecticide | Cypermethrin 250.0 g/L | Registered | 50 mL | 2.08 mL |
Standak Top | Insecticide Fungicide | Pyraclostrobin 25 g/L + Thiophanate-methyl 225 g/L + Fipronil 250 g/L | Not registered | 360 mL | 0.69 mL |
Poquer | Herbicide | Clethodim 240g/L | Registered | 400 mL | 2.77 mL |
Fusilade 250 EW | Herbicide | fluazifop-butyl 250 g/L | Registered | 700 mL | 4.84 mL |
Gamit 360 CS | Herbicide | Clomazone 360.0 g/L | Registered | 2,000 mL | 13.83 mL |
Callisto | Herbicide | Mesotrione 480.0 g/L | Not registered | 400 mL | 2.77 mL |
Compatibility was assessed based on the adaptation of the IOBC/WPRS protocol, proposed by Vainio (1992). For this purpose, product mixtures were prepared at twice the dose indicated by the manufacturer (Table 2). Then, 1 mL of this solution was added to flat-bottomed glass tubes, and 1 mL of the nematode suspension in distilled water was also added, at a concentration of 2000 IJs/mL, resulting in 2 ml of solution at the concentration indicated by the manufacturer with 1,000 IJs/mL. A control treatment was carried out using only the suspension of EPNs in distilled water at a concentration of 1,000 IJs/mL. Each treatment was repeated five times and the experiment was conducted in a completely randomized design. The tubes were kept in a climatic chamber at 22 ± 1°C and 14 hr of photophase, for 48 hr, when the viability and infectivity were evaluated.
The viability assessment was carried out after 48 hr. After shaking the suspension, five 50 µL aliquots were removed from each tube and individually transferred to Elisa well plate. The number of live and dead IJs was counted until the total of 100 IJs, considering dead individuals those who did not move when touched with a probe.
To assess the infectivity, 3 mL of distilled water were added to each tube, stirred, and left to decant for 30 min at 10°C in the refrigerator. Then 3 mL of the supernatant was discarded, and the procedure repeated three times to eliminate product residues. After the third wash, five 0.2 mL aliquots were removed from the suspension of each tube and applied individually in Petri dishes (9 cm in diameter), containing two filter papers and 10 last instar larvae of
All experiments were repeated in time. The data from the selection test, different concentrations, displacement, as well as the viability and infectivity of the compatibility test were subjected to analysis of variance, and if the assumptions for homoscedasticity and normality were met, the means were compared by the Tukey test (
Phytosanitary products were also classified based on the value of E%, based on the formula prepared by Peters and Poullot (2004), but modified in this work, since production values were not considered. Thus, E% was calculated using the formula:
And Rinf% refers to the reduction of infectivity in treatments, which was determined by the formula:
Based on the value of E%, the products were classified as: 1 – harmless (< 30%); 2 – slightly toxic (30-79%); moderately toxic (80-99%), and 4 – toxic (>99%).
All isolates evaluated showed pathogenicity and caused confirmed infection in females of
Mortality (%) of
Isolate | Species | % Mortality |
---|---|---|
Control | – | 0.0 ± 0.0 aa |
RSC05 |
|
46.0 ± 6.0 bc |
IBCB-n46 |
|
48.5 ± 10.3 bc |
IBCB-n44 |
|
48.9 ± 20.1 bc |
IBCB-n05 |
|
52.5 ± 6.5 bc |
JPM4 |
|
54.0 ± 8.7 bc |
JPM3 |
|
54.7 ± 6.3 bc |
RSC 03 |
|
59.4 ± 7.4 cd |
GL (Alho) |
|
61.0 ± 14.8 cd |
IBCB-n40 |
|
84.0 ± 8.1 de |
NEPET11 |
|
93.8 ± 4.1 e |
The isolate of the species
In the present study, the isolates that showed promise for the control of
Virulence variability for isolates from different species of nematodes on the same host was expected (Lewis et al., 2006), and has already been reported in the work of Ferreira et al. (2015) for
Intraspecific variation was also expected (Simoes and Rosa, 1996) and reported for other species of nematodes (Campos-Herrera and Gutierrez, 2009). Although less clear as to its origin, this variation may be related to the characteristics of the isolate, biotic, and abiotic factors of the soil from which they were found, and the host species with which they had contact during the evolutionary process (Shapiro-Ilan et al., 2006).
A factor that may have contributed to the differences in virulence observed in the present study is that the
Both isolates of
Compatibility of the entomopathogenic nematode
Data | Treatment | Viability (%)a | Infectivity (%) | MC% | Rinf% | E% | Classificationb |
---|---|---|---|---|---|---|---|
28/05/2015 | Control | 92.2 ± 0.6 aa | 92 ± 3.7 a | – | – | – | – |
Curyom 550 EC | 90.8 ± 1.2 a | 8 ± 3.7 b | 1.52 | 91.30 | 92.82 | Moderately toxic | |
Poquer | 79.8 ± 3.1 b | 90 ± 6.3 a | 13.45 | 2.17 | 15.62 | Innocuous | |
20/06/2015 | Control | 99.4 ± 0.2 a | 86 ± 5.1 a | – | – | – | – |
Fusilade 250 EW | 99.2 ± 0.4 a | 62 ± 8.6 a | 0.20 | 27.91 | 28.11 | Innocuous | |
Cipermetrina 250 EC | 97.4 ± 0.6 b | 80 ± 8.9 a | 2.01 | 6.98 | 8.99 | Innocuous | |
Gamit 360 CS | 99.0 ± 0.3ab | 74 ± 9.3 a | 0.40 | 13.95 | 14.36 | Innocuous | |
Nomolt® 150 | 99.2 ± 0.2 a | 66 ± 6.8 a | 0.20 | 23.26 | 23.46 | Innocuous | |
Callisto | 98.0 ± 0.5 ab | 90 ± 5.5 a | 1.4 | 0.00 | 1.41 | Innocuous | |
24/07/2015 | Control | 92.8 ± 0.9 a | 86 ± 2.4 a | – | – | – | – |
Tiger 100 EC | 76.6 ± 2.5 b | 76 ± 2.4 a | 17.46 | 11.63 | 29.08 | Innocuous | |
Standak top® | 87.2 ± 1.7 a | 72 ± 4.9 a | 6.03 | 16.28 | 22.31 | Innocuous | |
Actara 250 WG | 70.8 ± 1.9 b | 86 ± 5.1 a | 23.71 | 0.00 | 23.71 | Innocuous | |
Gaucho FS | 71.2 ± 2.5 b | 86 ± 2.4 a | 23.28 | 0.00 | 23.28 | Innocuous |
The NEPET11 isolate was more virulent, providing the same control index at the lowest concentration (25 IJs/cm2) as the IBCBn-40 isolate at the highest concentration used (200 IJscm2) (Fig. 1). In a similar study, Guide et al. (2016) observed that the NEPET11 isolate caused 100% mortality in
The high virulence of heterorhabditids on mealybugs was also reported by Ferreira et al. (2015), where the authors observed that the
Lethal concentration is directly linked to the virulence of the isolate, and may be due to factors related to the nematode and the host, such as the search strategy of the isolate, the way it penetrates and causes infection, as well as its ability to circumvent the host’s immune system. Obtaining effective control at low concentrations is important, as it may lower costs for production and application of the isolate in biological control programs (Ferreira et al., 2015; Guide et al., 2016).
On the other hand, low concentrations obtained in the laboratory may not be as efficient in the field, where several other factors can interfere with the nematode’s action. Thus, experiments in field conditions must be carried out to confirm these values.
In the vertical dispersal test, significant mortality of scale insects up to a depth of 20 cm was observed (Fig. 2). There was a significant reduction in mortality at depths of 10 and 20 cm (70 and 45%, respectively) in relation to the column surface, where mortality reached 90%. However, the dispersal of the EPNs up to 20 cm in the sand column, confirmed by the mortality of scale insects at this depth, means a favorable dispersal for possible use in the field, since the roots of the cassava culture are located mainly in the arable part of the soil, between 0 and 40 cm depth (Oliveira et al., 2001; Pequeno et al., 2007; Souza et al., 1994).
For the horizontal dispersal test, mortality was observed in all evaluated positions, regardless of their disposition (Fig. 3), with values above 85%.
Guide et al. (2016) also observed vertical dispersal of the NEPET11 and RSC05 isolates in search on
In the field, the IJs move due to olfactory stimuli, released by the host (CO2 or other components secreted by the host) (Hallem et al., 2011; Dillman et al., 2012), volatile substances secreted by plants (Shapiro-Ilan et al., 2017), or even by identifying the host’s physical vibrations (Torr et al., 2004). Abiotic factors, such as temperature, humidity, pH, and soil type, can also interfere (Stuart et al., 2015). Although in this work, dispersal was observed only up to 20 cm, longer permanence in the environment or the presence of the host insect can serve as a stimulus for dispersal to greater depths.
The compatibility test between the phytosanitary products and the NEPET11 isolate was performed at three different times, according to the dates recorded in Table 4. The products that reduced nematode viability were: Poquer® (Clethodim) (20.2%), Tiger 100EC® (Pyriproxyfen) (23.4%), Actara 250WG® (Thiamethoxam) (29.2%), and Gaucho FS® (Imidacloprid) (28.8%) (Table 4). The other tested products did not significantly affect the viability and presented values close to the controls.
The assessment of infectivity after exposure of the NEPET11 isolate to the products indicated that the insecticide Curyom reduced the percentage of infectivity on
Andaló et al. (2004b) evaluated the compatibility of phytosanitary products recommended for coffee cultivation with entomopathogenic nematodes (
Tavares et al. (2009) performed a compatibility test of
Some phytosanitary products from the chemical group of organophosphates, in which the Curyom® product belongs, are recommended to control phytonematodes (AGROFIT, 2020). The active ingredient of this insecticide, profenofos, acts mainly by inhibiting acetylcholinesterase (enzyme that breaks down acetylcholine) (Santos et al., 2007), which affects the motor coordination of the organism, inhibiting the feeding and movement of contaminated individuals, which may have affected the ability of the NEPET11 nematode to cause infection in the host. Similar results have been reported for the isolate IBCB-n40, which after exposure to the insecticide carbofuran, exhibited no effect on viability, but its infectivity decreased (Bortoluzzi et al., 2013).
Thus, at the end of the compatibility test in this work, and according to the calculation of E%, only the product Curyom 550 EC was considered moderately toxic, with all the others being harmless to the NEPET11 nematode.
In addition to virulence data, other factors must be taken into account when aiming at the use of an entomopathogen in pest control programs, such as data on the nematode search habit, as well as the possibility of integration with the pesticides used in the culture, through compatibility studies between the EPNs and products used in agriculture. EPNs are generally compatible with some chemicals, which allows for joint use with chemical control methods (Andaló et al., 2004b; Negrisoli et al., 2008a, b).
In addition, the use of native isolates from regions near the pest area may be better adapted to the same conditions as the host, having developed specificity, thus, exerting greater virulence and pathogenicity even at lower concentrations, which is commercially interesting (Alves and Moino, 2009; Ferreira et al., 2015). These nematodes are also more advantageous from the environmental point of view compared to exotic species (Dolinski and Moino, 2006).
The NEPET11 isolate of