Antibiosis resistance of soybean genotypes to Diabrotica speciosa ( Germar , 1824 ) ( Coleoptera : Chrysomelidae )

This research aimed to discriminate antibiosis-type resistant soybean genotypes to Diabrotica speciosa (Germar, 1824). Six soybean genotypes were used, according to previous selection, which were: IAC 100, BRSGO 8360, IGRA RA 626 RR, DM 339, PI 227687 and PI 274454. A completely randomized design was adopted, with six treatments and 10 replicates. Each replicate was constituted by 10 soybean plants and 10 D. speciosa larvae, in the initial infestation, doubling the number of plants in the transferring, 10 days after the initial infestation, using fine-grained vermiculite as substrate. In this experiment, the following characters from D. speciosa were assessed: Number of males, females and total of emerged insects, total survival (percentage), sex ratio, male and female weight (mg), larva to adult period and longevity (without food). The genotypes DM 339, PI 274454, and PI 227687 show antibiosis resistance against D. speciosa.


INTRODUCTION
The Diabrotica genus (Chevrolat, 1844) (Coleoptera: Chrysomelidae) includes some of the most harmful pests that occur in the whole America (Cabrera Walsh, 2003).In the last decades, the relevance of the species from this genus has increased in consequence of serious injuries caused by native species on plants from different crops in the American continent (Krysan, 1986;Tollefson, 1998).Diabrotica speciosa (Germar, 1824) is already established in many Brazilian states, and has economic expression as it is a polyphagous pest by causing losses to crops such as soybeans (Glycine max [L.] Merril), beans (Phaseolus vulgaris L.), maize (Zea mays L.), peanuts (Arachis hypogaea L.) and potatoes (Solanum tuberosum L.), between others, due to the injuries caused on the plant.This species is also a vector of plant pathogens, primarily viruses (Laumann et al., 2003).
D. speciosa has a holometabolous life cycle (Milanez and Parra, 2000).Females lay eggs in the soil, around the plants.The incubation period ranges from six to eight days.The larval stage goes through three instars, and its mean duration period is 18 days.After completing the larval stage, the insects are directed to the soil, where they build a pupal chamber to develop into pre-pupae and pupae.The pre-pupae mean duration period is five days, and the pupal stage is seven days.The life cycle varies from 24 to 40 days (Ávila and Parra, 2001;Milanez and Parra, 2000).
The larvae of this chrysomelid feed and develop on the roots and tubers of several crops (Milanez and Parra, 2000).The feeding damage can cause developmental delay and increase in the susceptibility of the plants to lodging (Marques et al., 1999).Conversely, the adults feed on aerial parts of plants, such as leaves, stems, fruits, and pollen of crops and wild plants (Gassen, 1989).
This insect species is one of the most abundant and harmful pests in Brazilian crops (Christensen, 1942;EPPO, 2005) and presents a hazard to the European continent, as well other species from North America (EPPO, 2005).
To control this pest, the main additional problem is the environmental risk associated to the chemical control by synthetic insecticides.Excessive insecticide applications lead to resistance in pests, human health risks and hazards for natural enemies and pollinators (Ávila and Nakano, 2000).This modality demands relatively higher amounts of active ingredient per area, and hence increases the costs and it may contaminate the groundwater, mainly in arenaceous soils (Pereira et al., 2005).
In modern agriculture, host plant resistance is an integral component, if not the basis, of arthropod pest regulation in integrated pest management (IPM) programs (Panda and Kush, 1995;Smith, 2005;Stout, 2007).The plant resistance category known by antibiosis occurs when the negative effects from a resistant plant affect the biology of an arthropod, when it uses the plant to feed upon.The antibiotic effects from a resistant plant vary from moderate to lethal, and may result due to chemical and morphological mechanisms (Smith, 2005).
Introductions of the soybeans lines PI 171451, PI 227658 and PI 229358 have been used since the beginning of the 1970's decade as resistant sources to insects which feed on leaves, as D. speciosa (Rezende and Miranda, 1980).
Thus, this research aimed to discriminate antibiosistype resistant soybean genotypes to D. speciosa.

MATERIALS AND METHODS
The experiment was conducted at the Laboratório de Resistência de Plantas a Insetos, from the Departamento de Fitossanidade of the Faculdade de Ciências Agrárias e Veterinárias -FCAV/UNESP, under temperature conditions of 25 ± 2°C, relative humidity of 70 ± 10% and a photophase of 12 h.

Insects breeding
The D. speciosa rearing method used for the elaboration of this experiment was carried out according to the methodology proposed by Ávila et al. (2000).The adult insects were collected in a bean crop.
To obtain D. speciosa eggs, Petri dishes (14 cm in diameter × 2 cm in height) were used, containing in their interior moistened cotton and, on this, black gauze as substrate for oviposition.The eggs were removed from the oviposition substrate by rinsing the gauze in running water into synthetic polyester fabric of 900 cm² (mesh size of 0.03 × 0.03 mm), where the eggs were retained.In order to avoid the contamination by fungi and other microorganisms during the incubation period, the eggs were treated with copper sulfate solution (CuSO4) at 1%, per two minutes, and later, they were transferred into Petri dishes (9 cm in diameter and 1 cm in height), lined with moistened filter paper (Ávila et al., 2000).
For the rearing, an initial infestation of neonate larvae was performed in plastic containers (17 cm in diameter × 9 cm in height).First, the containers were filled with 40 g of fine-grained vermiculite moistened with 50 g of deionized water.Afterwards, 70 plants of AL-Piratininga variety were placed on the moistened vermiculite, and subsequently 70 larvae were put on the maize roots for feeding and developing.Ten days later, the larvae were transferred to larger plastic containers (27 cm in length × 16 cm in width × 9.5 cm in height).The amounts of deionized water, vermiculite, and plants were doubled, aiming to provide healthy plants and larger space for the developing of larvae and pupae of D. speciosa, until the adults emergence.The maize seeds were treated with the fungicide carbendazim + thiram (Derosal Plus ® ), applying 200 ml of the commercial product to 100 kg of seeds.
The D. speciosa adults were fed on bean leaves of IAC Carioca-Tybatã variety, which were placed in glass cages (40 cm long × 30 cm high × 30 cm wide)

Bioassay
For the experiment conduction, the sequence and rearing methodology formerly reported were followed, using, although, plants from the studied soybean genotypes, as follows: IAC 100, BRSGO 8360 (susceptible), IGRA RA 626 RR (resistant), DM 339, PI 227687 (resistant) and PI 274454 (resistant), which were screened from the results obtained in a previous feeding preference test for adults (Costa et al., 2012).The soybean seeds were treated with the fungicide carbendazim + thiram (Derosal Plus ® ), by applying 200 ml of the commercial product to 100 kg of seeds.
A completely randomized design was used, with six treatments (genotypes) and 10 replications.Each replication was constituted by 10 soybean plants and 10 D. speciosa larvae, in the initial infestation, totaling 100 plants and 100 larvae per treatment.On the transferring occasion (10 days after the initial infestation), the surviving larvae were relocated to another container, with similar quantities of vermiculite and water, as previously mentioned, but only the number of plants were doubled, in order to provide greater food amount (soybean roots) for the insects in the immature phase.For both process, initial infestation and transferring, plastic containers of 12 cm in diameter × 9.5 cm in height were used.
The following characters of D. speciosa were evaluated in the experiment: Number of males, females and total of emerged insects, survival (percentage), sex ratio, weight (mg) of males and females, using a precision analytical balance (model AS200S, Florham Park, NJ), larva to adult period, and longevity (without food).

Statistical analysis
Data obtained in these experiments were submitted to normality  (Shapiro-Wilk) and homocedasticity (Bartlett) tests (Silva and Azevedo, 2006).Considering that they are non-normal variables, which could not be normalized by transformations, the Kruskal-Wallis' non-parametric test (H statistic) was used.Analyses were carried out at the P < 0.05 level of probability.

RESULTS
Differences were observed amid the soybean genotypes in the following biological variables: Number of males, females and total of emerged insects (Table 1), total survival and longevity (Table 2), larva to adult period and females weight (Table 3).Regarding the number of emerged males (H = 18.66; df = 5; P < 0.01), the highest values were found for the genotypes BRSGO 8360, 1.10, and IGRA RA 626 RR, 1.30, whilst for the genotype DM 339 the occurrence of individuals of this sex was not recorded.Concerning the other variables cited, the genotype BRSGO 8360 highlighted with the highest values.Analyzing the number of emerged females (H = 18.62; df = 5; P < 0.01), BRSGO 8360 stood out with the highest value, 1.90, differing from all the other genotypes.No one female emerged from the genotype PI 274454.
Regarding the total number of emerged insects (H = 22.48; df = 5; P < 0.01), the genotypes BRSGO 8360, IGRA RA 626 RR, and IAC 100 held the highest mean numbers, with 3.10, 2.30, and 1.70, respectively, differing from the genotypes DM 339, PI 274454, and PI 227687, which showed 0.20, 0.30, and 0.40, respectively.These data can be expressed in total survival (%) (Table 2), in which the genotypes were divided similarly into two groups.It is important to note that the total survival on BRSGO 8360 (31.00%) was 15.5 times higher than on DM 339 (2.00%).

DISCUSSION
The results obtained in this experiment demonstrated, in general, that the genotypes DM 339, PI 227687 and PI 274454 were the least suitable for D. speciosa development, by expressing resistance by antixenosis to the pest, while for the genotypes IAC 100, BRSGO 8360 and IGRA RA 626 RR, favorable results for the insect development were found.Ávila and Parra (2002), while studying the D. speciosa development on different genotypes, concluded that under field conditions, in the absence of a preferred host, the larvae of this species may use soybean plants as an alternative host for its feeding and breeding.In Brazil, Corseuil et al. (1974) reported that larvae of D. speciosa feed on soybean plants roots.Hoffmann-Campo et al. (2000) mentioned that this pest has been causing preoccupation to soybean growers from the west and southeast regions of Paraná state.According to Cabrera Walsh (2003), D. speciosa larvae developed well on maize, peanuts, and soybean roots.
In the 1990 decade beginning, a strain of Diabrotica virgifera virgifera (LeConte) became established in the USA Corn Belt, adapting itself to the maize-soybean crop rotation by the oviposition in soybean fields, which must be rotated to maize crop in the next year (Gray et al., 1998).However, the current research demonstrates the relevance of studying the host plant resistance, by detecting genotypes which highlight regarding the average, that is, express resistance.Thus, this type of experiment would be very important to the subsequent utilization of resistant genotypes in areas which the cultivation of different crops is carried out, such as in Brazil, in order to reduce the pest density in the off season.The genotype DM 339, as verified in the current research, is a resistant material, and as it is a commercial cultivar, perhaps it is an option in the attempt to decrease the D. speciosa population level, when, for instance, the maize-soybean crop rotation is necessary.It is relevant yet to consider that this pest species is multivoltine, and as mentioned before, it can feed on soybean roots.Ávila and Parra (2002), when evaluating the D. speciosa development on different hosts, concluded that the insects' survival which fed upon soybean roots from the genotype IAC-8 was 30.1%, very close to the rate found on BRSGO 8360 in our study (31.0%).However, when comparing different hosts on the development of an insect, it is important to know the resistance of the studied genotypes, having in order the dissimilarity detected among the genotypes in this work, where the survival values ranged from 2.0 to 31.0%.Dunbar (2011) assessed the soybean varieties effects on D. v. virgifera, and concluded there were no differences amidst the varieties with the genes rag1, rag1/rag3 and a susceptible isoline from rag1 variety on the survivorship, eggs production or leaf intake of the soybean varieties, and despite these varieties are resistant to Aphis glycines Matsumura, the same does not seem to impose natural selection against D. v. virgifera resistance in the maize-soybean crop rotation.The same authors affirm the varieties utilization which reduce the D. v. virgifera fitness, may help in the delaying of the pest insect resistance evolution.On the other hand, this research revealed genotypes expressing resistance, emphasizing the importance of the study on materials from different origins, comparing lines and cultivars, for instance.

Table 1 .
Number (mean ± standard error) of males, females and total of emerged insects of Diabrotica speciosa per container fed on different soybean genotypes.

Table 2 .
Total survival (%), sex ratio and longevity (mean ± standard error) of D. speciosa individuals fed on different soybean genotypes.

Table 3 .
Larva to adult period (days), and male and female weight (mg) (mean ± standard error) of D. speciosa fed on different soybean genotypes.