Control of Mahanarva fimbriolata ( Stål ) ( Hemiptera : Cercopidae ) with entomopathogenic fungus and insecticides using two sampling methods on sugarcane fields

The sampling of M. fimbriolata aims to estimate the population level and optimum timing for spittlebug control in the sugarcane fields. Thus, this study was conducted to determine the cost and efficiency of Metarhizium anisopliae (Hypocreales: Clavicipitaceae) and insecticides utilized in M. fimbriolata control using two sampling techniques. The experiments were performed between November 2012 and April 2013, in a sugarcane field. In each experiment, apart from the control, treatments included Thiamethoxan (250 g ha), Imidacloprid (700 g ha), PL 43 (M. anisopliae 2.0×10 con mL), ESALQ E9 (M. anisopliae -2.1×10 con mL) and IBCB 425 (M. anisopliae 1.4×10 con mL). In the first experiment, the M. fimbriolata nymphs and adults were monitored, while on the other experiment, the nymphs sampled were the small, medium, large, and the adults of M. fimbriolata with objective was to identify the timing of the application of treatments. In general, by monitoring the small, medium and large nymphs and the adult spittlebugs, the timing of the application was optimized, which increased the efficiency of M. fimbriolata control.

In order to reduce the M. fimbriolata populations (Dinardo-Miranda et al., 2004a, b;Loureiro et al., 2005;Cuarán et al., 2012) insecticides and entomopathogenic fungi like Metarhizium anisopliae (Metschnikoff, 1879;Sorokin, 1883) (Hypocreales: Clavicipitaceae) are used to protect the sugarcane plantations.Control of the spittlebug begins with monitoring the pest, immediately after the first spring rains (Almeida et al., 2007).Using the conventional sampling method, the M. fimbriolata nymphs and adults are monitored at 3 points per hectare on 2 linear feet of row sugarcane planting and an ideal frequency of 15 days (Mendonça, 2005;Dinardo-Miranda et al., 2007).
However, the sampling of the nymphs and adults can impede the identification of the end of the life cycle as well as the timing of control of M. fimbriolata (Kassab et al., 2012).Highly infested areas could render the sample data unreliable, while regular rainfall could trigger the resurgence of the spittlebug populations, negating the effects of the insecticide and/or bioinsecticide applications during the control of M. fimbriolata.Therefore, Kassab et al. (2012) suggested modifying the M. fimbriolata sampling by monitoring the small nymphs (up to 5 mm), medium (6-10 mm) and large ones (over 10 mm) as well as the adult spittlebugs.This proposal enables the observation of the end of the cycle of M. fimbriolata generations and the best timing at which to control this pest, the period when the large nymphs are more numerous than the small and medium nymphs.It is at this time, which the M. fimbriolata adults can also be sampled in the pest population, which may indicate that eggs of the spittlebug had favorable to emergence of this insect.
Regardless, the new sampling method proposed for M. fimbriolata (Kassab et al., 2012) is yet to be compared with the conventional method (Mendonça, 2005;Dinardo-Miranda et al., 2007) and this indicates the importance of understanding the efficiency of the entomopathogenic fungi and insecticides in the control of M. fimbriolata by employing both these methods.
The aim of this study was evaluating the cost and efficiency of M. anisopliae and the insecticides used in controlling M. fimbriolata employing two sampling methods.

MATERIALS AND METHODS
A sugarcane field owned by Energética Santa Helena Ltda., a company in Nova Andradina, Mato Grosso do Sul State, Brazil was the site where the experiments were conducted between November 2012 and April 2013.The experimental area (S 22º13´58´´, W 53º20´34´´ and 380 m asl) was planted with the SP81-3250 variety of sugarcane, with no defects in the sprouting plants.
In this study, we followed the randomized blocks design (RBD) with six treatments and four replications of each, that is, with and without the conventional sampling of M. fimbriolata.The plots included 10 lines of sugarcane spaced 1.4 m apart and 10 m long, an area of 140 m 2 .
Using the conventional sampling method, the experiment with M. fimbriolata was performed with the weekly monitoring of the spittlebug nymphs and adults (Mendonça, 2005;Dinardo-Miranda et al., 2007).The first application of control for M. fimbriolata was done on 23 November, 2012 and the second on 15 Janand, 2013, when the degree of control of M. fimbriolata was achieved (Mendonça, 2005).To conduct the experiment with the proposed new sampling method, weekly sampling was done of the small, medium and large nymphs and adult individuals of M. fimbriolata to identify the end of the cycle of the spittlebug generations (Kassab et al., 2012) which occurred on 15 January 2013.
Coastal sprayers calibrated for a flow rate of 150 L ha −1 (Mendonça, 2005) were used and the insecticide was directed at the stump bases so that 30% of the spray volume reached the stems and 70% reached the sugarcane plant roots (Loureiro et al., 2005).The surfactant Tween ® (0.01% polysorbate 80) was used to treat the fungal suspensions.
The M. fimbriolata nymphs were sampled every two weeks, up to 60 days after treatment (DAT) in the two linear meters of the furrowplanted sugarcane, in each plot, in the experiments using the two sampling methods.The M. fimbriolata nymphs found on the basal sugarcane internodes were counted after removing the residual straw.
Climatic conditions including average temperature, relative humidity and rainfall were represented with data from INMET (Instituto Nacional de Meteorologia) (Figure 1) to determine the relationship between the abiotic factors and M. fimbriolata infestation.The total value of recoverable sugar (TRS) according to Landell et al. (1999) was obtained from the sugarcane stalks randomly selected on 15 April 2013.
The tonne per ha value (TRS × TRS quote), estimated a yield per ha [68 tonnes (productivity of Mato Grosso do Sul State (Unica, 2013) × value of a tonne per ha], maintenance costs of the sugarcane (MCS) without the product and the cost involved in the application of M. fimbriolata control (Udop, 2013), cost control (including product and application expenses) and earnings per ha (estimated production per ha − MCS) were calculated in dollars (US$) for both the experiments.The expenses for the services outsourced and purchase of products to improve the sugarcane plantation were obtained from consulting firms and the UDOP agricultural database of 2013 (Table 1).
The population data for the M. fimbriolata nymphs recorded between 23 November 2012 and 15 January 2013 were subjected to the analysis of variance and the means were compared using the Scott-Knott test at 5% probability.Further, using Abbott's formula (Abbott, 1925) treatment efficiency was calculated and the means were compared by the Scott-Knott test at 5% probability.The average of the M. fimbriolata nymphs and the efficiency control (Abbott, 1925), post the 15 January 2013 treatment in the experiments following the two sampling methods were compared by the analysis of variance (ANOVA -F TEST) at 5% probability.

Experiment with conventional sampling: between 23 November 2012 and 15 January 2013
The number of nymphs and efficiency of M. fimbriolata

Conventional sampling × new sampling method: Between 15 January 2013 and 17 March 2013
There was no difference in the number of nymphs of M. fimbriolata between the treatments during the experiments, both with and without the conventional sampling, at 15 and 30 DAT (Table 3).The control efficiency of the M. fimbriolata nymphs did not differ between treatments with and without conventional sampling in the assessments at 15 and 30 DAT (Figures 3A and B).
At 45 and 60 DAT, all the treatments utilizing the new sampling method showed significantly less infestation (Table 3).The treatment efficiency of the new sampling method was higher when compared with the conventional method (Figures 3C and D).Imidacloprid insecticide and the isolate IBCB 425 of M. anisopliae were which achieved the highest efficiencies of M. fimbriolata control at 45 and 60 DAT (Figures 3C and D).The increase in the number of M. fimbriolata nymphs parasitized by M. anisopliae (Figure 4), at 45 and 60 DAT, was due to the higher rainfall (mm), temperature (°C) and relative humidity (%) (Figure 1).
The total recoverable sugar (TRS) values, which included the expenses incurred per tonne and the earnings received per ha were higher in all the treatments employing the new sampling method (Table 4).The values regarding the acquisition and application of the products in the treatments with the conventional method were higher than in the other method, because two applications had been done for the control of M. fimbriolata, and therefore, lesser profits were made with the conventional method (Table 4).

Experiment with conventional sampling: Between 23 November 2012 and 15 January 2013
The lower variation recorded regarding the number of M. fimbriolata nymphs following the conventional sampling method, for 15 DAT, 30 DAT and 45 DAT can be related to the monitoring method used for this insect.In this method, the application of the insecticides and entomopathogenic fungi can occur in the initial stage of the first cycle of the spittlebug generations, that is, September/October (Almeida et al., 2007).During this  Thiamethoxan, Imidacloprid and M. anisopliae (Hypocreales: Clavicipitaceae) using the conventional method of sampling from 23 November 2013 to 15 January 2013.Means followed by the same letter, in each bar, were compared using the Scott-Knott test at 5% probability period, most of the M. fimbriolata diapausing eggs which are not hatched and due to the greater volume and regularity of rainfall between November and December can increase the possibility of reapplication of these products to control this pest (Kassab et al., 2012).At 60 DAT, a lesser degree of infestations of the M.
fimbriolata nymphs was obtained with Imidacloprid and thiamethoxan possibly as a result of the action of these insecticides, which may have contributed to a more efficient control of the spittlebug.The chemical molecule has a greater residual effect than that of the entomopathogenic fungi, which implies that this product     can persist longer on the crop and more effectively control this pest (Dinardo-Miranda et al., 2004b;Carvalho et al., 2011).
The entomopathogenic fungi, however, lose their viability after the application which could influence the effectiveness of the treatments on the isolates of M. anisopliae (Lopes et al., 2011;Guerrero-Guerra et al., 2013).
There were fewer M. fimbriolata nymphs parasitized by M. anisopliae in the experiment following the conventional sampling method, which can be explained by the timing of the treatment application.The conventional sampling method not considers the biotic potential of the area (Kassab et al., 2012) and the insecticide and/or bioinsecticide application in M. fimbriolata control occurs in the low density nymphs (Mendonça, 2005).This simply means that if, in the first application, during spittlebug control the temperature and humidity favor the development of the M. fimbriolata nymphs (Freire et al., 1968), new applications of biological and chemical insecticides will be necessary.This is similar to what was observed in this experiment with the first application in M. fimbriolata control on 22 Nov. 2012, followed by the second application on 15 January, 2013.

Conventional sampling × new sampling method: between 15 January 2013 and 17 March 2013
The number of M. fimbriolata nymphs did not differ between the treatments followed in the two sampling methods, at 15 and 30 DAT.This indicates that the entomopathogenic fungi and insecticides may have a protracted effect in the field (Dinardo-Miranda et al., 2004a;Loureiro et al., 2005;Tiago et al., 2012).However, although generalizations cannot be made, the cumulative effect is controlled by the weather, the active constituent of the insecticide, the chemical mode of action, and the M. anisopliae isolated to control M. fimbriolata (Dinardo-Miranda et al., 2004a;Loureiro et al., 2005).On other hand, the genetic constitution of the pest population (Quinelato et al., 2012), adaptations and mechanisms of insect resistance (Dubovskiy et al., 2013) may influence the efficiency of the control techniques.
At 45 and 60 DAT, the treatments utilizing the new sampling technique were significantly less infested and revealed greater efficiency in the control of M. fimbriolata.The insecticide Imidacloprid and the isolate of M. anisopliae (IBCB 425) showed the highest control efficiency.This result was anticipated, as the late application of the insecticides to control the M. fimbriolata reduced the spittlebug infestations and increased the sugarcane productivity (Dinardo-Miranda et al., 2004a;Madaleno et al., 2008).Furthermore, the application occurred towards the end of the cycle of M. fimbriolata generations and, it is now accepted, that the normal and diapausing eggs of this pest, experienced conditions suitable for the emergence of nymphs which, in turn, may have enabled greater control efficiency (Kassab et al., 2012).
The number of M. fimbriolata of nymphs parasitized by M. anisopliae rose with the increase in rainfall (mm), temperature (°C) and relative humidity, which can be explained by the action of the climate-dependent entomopathogenic fungi (Almeida et al., 2007).The entomopathogenic fungi can also increase their density in the crop by infecting healthy individuals (Bruck, 2005, Bruck andDonahu 2007).Furthermore, the insects destroyed by M. anisopliae remain in the field, which can reduce the possibility of the resurgence of these pests (Guerrero-Guerra et al., 2013).
The value of the total recoverable sugar (TRS), the expenses per tonne and the earnings per ha were higher in all the treatments which employed the new sampling method.Moreover, the isolates PL 43 and IBCB 425 obtained a higher TRS value, which may be a result of the action of the Metarhizium ssp. to translocate the nitrogen of the parasitized insect to the plants (Behie et al., 2012).Besides, plant age can also influence the sugarcane productivity (Dinardo-Miranda et al., 2008).Thus, the plants at a more advanced developmental stage may show a higher TRS yield.The TRS value was estimated using 8-month-old plants, although it may be higher for the older individuals.
Careful monitoring of the small, medium and large nymphs and the M. fimbriolata adults can optimize the time of application and raise the efficiency of spittlebug control.Besides, from this study, monitoring the M. fimbriolata populations is best done after the first spring rains, following the diapause period of the spittlebug eggs.Areas with a history of M. fimbriolata infestations should be given priority in the monitoring programs of this pest and the timing of the insecticide application must be synchronized with the end of the spittlebug lifecycle, thus, lowering the likelihood of reapplication of insecticides in its control.

Conclusion
The use of M. anisopliae and insecticides along with monitoring the small, medium and large nymphs and adult M. fimbriolata is the most suitable method to control the spittlebug populations by providing greater efficiency and lower cost per hectare.

Figure 2 .
Figure 2. Efficiencies in control of M. fimbriolata (Hemiptera: Cercopidae) nymphs in treatments with Thiamethoxan, Imidacloprid and M. anisopliae (Hypocreales: Clavicipitaceae) using the conventional method of sampling from 23 November 2013 to 15 January 2013.Means followed by the same letter, in each bar, were compared using the Scott-Knott test at 5% probability

Figure 3 .
Figure 3.Comparison of the efficiency of control of Mahanarva fimbriolata (Hemiptera: Cercopidae) nymphs utilizing the conventional method and new proposal of sampling from 15 Jan. 2013 to 17 Mar.2013.Means followed by the same letter, in each bar, were compared by the F test at 5%.

Table 1 .
Maintenance costs of sugarcane (MCS) per ha.
control per treatment did not differ in the ratings of 15 DAT, 30 DAT and 45 DAT (Table2 and Figures2A, B and C).At 60 DAT, the treatments with the fungus M.

Table 3 .
Comparative infestation of the Mahanarva fimbriolata nymphs (Hemiptera: Cercopidae) between experiments with the conventional method and the new sampling proposal from 15 Jan. 2013 to 17 Mar.2013.
b Means followed by the same letter, in each line, were compared using F test at 5%; DAT, Number of days after treatment; AC, sampling conventional; NPA, new sampling proposal; M.a., M. anisopliae.

Table 4 .
Total recoverable sugar (TRS), TRS cost (Cot.TRS), price per tonne (Tonne.Price.), production estimate per ha (Prod.ha), maintenance cost of sugarcane plantations (MCS), cost control of Mahanarva fimbriolata (Cost.Contr.), profit per ha (L.ha) in the treatments with insecticides and M. anisopliae (Hypocreales: Clavicipitaceae) utilizing the conventional and proposed new sampling methods with the values expressed in dollars (US$).