Insect Collection

L. zonatus adults were captured in an experimental plantation of J. curcas in the Development Center of Biotic Products from thr National Polytechnic Institute (CEPROBI) located in Barranca Honda, Yautepec Morelos (18° 49' north latitude and 99° 05' west longitude) at an elevation of 1071 masl with an annual average rainfall of 1050 mm and an average annual temperature of 22 °C (^{García, 1987}). Adults were also collected in bean and sorghum plots from the community of San Isidro Yautepec, near CEPROBI and in sorghum plots within the main municipality.

After the collection, insects were established in the laboratory from the National Plant Germplasm Bank (BANGEV) located in the Department of Plant Science at the Autonomous University of Chapingo (UACH) (Carretera Mexico-Texcoco km 38.5, Chapingo, Mexico state).

Insects were isolated in glass cages 30 x 30 x 40 cm and subjected to observation for five days to ensure that these were free of pathogens and parasites. The environment inside the cage was at a temperature of 25 to 30 ° C and relative humidity of 65 to 78%. Humidity was controlled with an electric humidifier Duracraft. Temperature was regulated by the heat emitted by 100 watts bulbs. Adults of L. zonatus were fed with sweet corn grains, fresh purging nut and fresh bean pods, previously washed with distilled water (^{Xiao and Fadamiro, 2009}).

Fungi characteristics

The strains used in bioassays (Table 1) were provided by the State Plant Health Committee of Guanajuato (CESAVEG) through MC Fernando Tamayo and reactivated at UACH, in culture medium solidified saborau dextrose agar (ADS), and kept in plastic Petri dishes at 25 °C for use in bioassays.

Evaluation of pathogenicity from three strain of B. bassiana

Pathogenicity and virulence were evaluated for the three strains and a control using a completely randomized design, each treatment had three replications and nine insects per repetition. For five days, adults of L. zonatus were kept isolated. At the end of the quarantine were immersed for three seconds in an aqueous suspension Tween 80 at 0.03% with 1 x 10⁸ conidia mL^-1 for each strain. The control consisted only in the immersion of the insects in water with Tween 80 at 0.03%. Immediately after immersion, insects were placed in Petri dishes with filter paper in order to remove excess of the suspension (^{Grimm and Guharay, 1998}). Later insects were placed in plastic containers of 500 cm³ according to each treatment and were fed with fresh fruits of J. curcas and fresh bean pods. During the bioassay, insects were kept in an incubator (Seedburo MP6-3000 B) at a temperature of 24 ± 1 °C with a range of relative humidity from 65 to 78% (^{Xiao and Fadamiro, 2009}).

Mortality assessment

Insect mortality was counted daily for 50 days and was adjusted through Abbott's formula. Dead insects were placed individually in Petri dishes with sterile filter paper moistened with distilled water and incubated for two weeks at 25 °C to promote the mycelia growth and production of conidia.

The sporulation percentage was determined and also the intensity thereof submerging sporulated insects in a solution Tween 80 at 0.03%. Stirred manually for 1 minute and then 10 µL aliquot was taken and diluted in 1 mL of the solution indicated at 0.03% and counted conidia using a Neubauer chamber.

Conidial germination

Conidial germination rate was determined by taking samples from the same suspension used in this bioassay (1 x 10⁸ conidia mL^-1) and placing aliquots of 10 µL in Petri dishes with ADS solidified and incubated for 24 h at 25 ± 1 °C . After this period, the number of germinated units of each 300 conidia was counted using a Neubauer chamber. This step was performed six times per strain.

Biological effectiveness of strain BB01

Once it was determined that the BB01 strain was the most pathogenic and virulent according to the results from the above assay, six treatments with three replicates, eight insects per replicate were established. Treatments consisted of five conidia concentrations (1 x 10⁴, 10⁵, 10⁶, 10⁷, 10⁸ conidia ml^-1) suspended in a solution Tween 80 at 0.03% and control which was only Tween 80 at 0.03%.

Conidia concentrations that cause 50% mortality, lethal dose 50 (LD50) was calculated taking into account the number of insects alive at the beginning and end of each treatment, including replications. The time in which 50% of the population died, lethal time 50 (LT50) was evaluated for both assays calculated through Probit analysis.

Data analysis

Data variables: 1) mortality rate; 2) sporulation percentage; 3) sporulation intensity; and 4) spore germination, were analyzed under a completely randomized design with an analysis of variance using SAS statistical software version 9.0 and a comparison of means using Tukey with a confidence interval of α= 0.05 for the first bioassay and LSD test with α= 0.06 for the second bioassay. The determination and evaluation of mortality rate, percentage and sporulation concentration were conducted in the same manner as in the first assay.

Pathogenicity of three strains of B bassiana

BB01 strain isolated from Melanoplus spp., was the strain with the highest mortality (85.71%) and higher percentage spore germination (99.28%), compared to the other two strains. In the variable percentage and sporulation concentration, there were no statistically significant differences (Table 2).

Figure 1 Sporulation of three Beauveria bassiana strains on adults of Leptoglossus zonatus. 

Beavueria bassiana has been used to regulate populations of different Coleopteran genera Diabrotica Colapis and Maecolapis (^{Sosa et al., 1994}) also reduces by up to 50 to 70% populations of Ixodes scapularis and Rhipicephalus sanguineus (^{Kirkland et al., 2004}). The application of B. bassiana in the soil reduces the emergence of adults of Diabrotica undecimpunctata, and root damage is less severe in soil treated with the fungus than in soils infested with the pest without conidial treatment (^{Krueger and Roberts, 1997}). ^{Grimm and Guharay (1998)} mentioned that can also be used as a biological control agent for L. zonatus and Pachycoris klugii, which are the two main pests of J. curcas in Nicaragua, Guatemala and Brazil.

It is possible that B. bassiana can exercise effective control in L. zonatus due to the high mortality rate obtained in this work, however, it must be taken into account that for an organism to be used as a biological control agent, is not enough a high mortality rate on the target organism, but must also achieve mortality fairly rapidly, so that lethal time 50, is a variable that becomes important. The BB01 strain evaluated in this study reached TL50 at 7.8 days, followed by BB02 and BB32 with 16.9 and 17.4 days, respectively (Table 3).

Figure 2 Lethal time 50 of three strains of Beauveria bassiana on adults of Leptoglossus zonatus. 

In relation to the speed of the fungus to have an effect on their host, Lezama et al., (1998) mention that some strains of B. bassiana exhibit varying virulence on Spodoptera frugiperda, with parasitism up to 90% in eggs and 100% in larvae, the most outstanding insulation wan an LT50 of 3.1 and 2.8 days in eggs and larvae of the same insect, and an LD50 of 2.4 x 10³ conidia mL^-1. Moreover, in bioassays performed on banana weevil Cosmopolites sordius, B. bassiana causes mortality of 63-97% at 35 days after exposure to the spores (^{Kaaya et al., 1993}). Given the above, it is possible to note that mortality and speed at which B. bassiana acts, depends on a) inoculated organism, b) fungus concentration of the application, c) state of development of target organism e) environmental conditions.

In bioassays on M. sanguinipes, strains of B. bassiana GK2016, "wild-type" (virulent) and GK2051, showed a LT50 of 5.8 and 7.8 days, respectively, although there were only two days apart in the LT50 value, strain GK2051 required 17 days to kill 90% of the population and never produced 100% mortality, while strain "wild type" GK2016, caused 00% death in 8-10 days (^{Kosir et al., 1991}). ^{Grimm and Guharay (1998)} report that when evaluating mortality on adults of L. zonatus inoculated with three different strains of B. bassiana, reached 50% mortality between 10 and 13 days after inoculation. It is possible to deduce that mortality of an organism, not only depends on its characteristics, but also depends on the characteristics of the strain used.

Considering the results shown, strain BB01 from B. bassiana evaluated in this experiment, is pathogenic and virulent on adults of L. zonatus than strains of the same fungus reported by ^{Grimm and Guharay (1998)}, for the control of the same insect.

Biological effectiveness of strain BB01 from B. bassiana

Due to higher mortality rate (85.7%), less time in days to reach LT50 (7.8 days) and the highest spore germination rate (99.28%), strain BB01 was used for the second bioassay. The suspension of 1 x 10⁸ conidia ml^-1 was the one with the highest values in variables mortality (100%), sporulation (79.16%) and sporulation concentration (19.53) compared to other treatments (Table 4).

In this regard, the mortality of larvae and adults of potato beetle Leptinotarsa decemlineata is related with applications in different doses (1 x 10⁴, 3 x 104 and 1 x 10⁵ conidia cm^2-1 leaf area) of B. bassiana (^{Fargues et al., 1994}). Moreover, ^{Grimm and Guharay (1998)}, by studying the effect of different strains of B. bassiana (1 x 10⁷ conidia mL^-1) on adults of L. zonatus, found mortalities of 88 to 99%. Different doses of strain BB01 from B. bassiana (Table 4), evaluated in this study, presented mortalities from 70% to 100%, the latter value was obtained in the concentration 1 x 10⁸ conidia mL^-1, so the mortality of L. zonatus is also related to the applied doses of the fungus.

Susceptibility and the relationship of fungi with the host are associated with nutrients present on insect body, which in turn are the medium for propagation, dispersion and persistence of such organisms (Gruwel and Eigenberg, 2007). Because of this, sporulation of the fungus in the insect's body is a dispersion medium and spread as seen in fire ant nests, Solenopsis invicta, where B. bassiana grows and sporulates (^{Pereira et al., 1993}), thus representing a potential biological control agent for this ant (^{Siebenelcher et al., 1992}). In this experiment, the dose 1 x 10⁸ conidia mL^-1 had the highest sporulation (79.167%) (Table 4), so the dispersibility of the fungus and infection to other insects, is greater.

LD50 of strain BB01 on adults of L. zonatus was 5.2 x 10⁷ conidia mL^-1 (Pr> ChiSq= 0.0021) (Table 5), so that LD50 is within the range of concentrations evaluated in this work.

In this regard doses of B. bassiana close to LD50 reduced the reproductive potential of S. lineatus, fertility and fecundity of C. suppressalis and egg development of leafhoppers in rice plants (^{Feng et al., 1994}). Moreover isolates of B. bassiana on larvae and adults of S. frugiperda, showed an LD50 of 2.4 x 10³ conidia mL^-1 and a TL50 of 3.8 days (^{Lezama et al., 1996}).

Figure 3 Lethal time 50 of three Beauveria bassiana strains on adults of Leptoglossus zonatus. 

The dose of 1 x 10⁸ conidia mL^-1 was the one with the lowest value in TL50 (5.9 days), so it is the dose that kills faster 50% of the population of L. zonatus (Table 6).

The lethal dose 50 of Diatrea sacharalis is related to the ingested spores dose, given the first deaths attributed to B. bassiana, which occurred at 7.7 days at dose 1 x 10⁸ conidia mL^-1, while for Tetranychus urtice with a dose 1 x 10⁸ conidia mL^-1, lethal dose 50 was at 3.6 days after inoculation (^{Batista et al., 2004}). Moreover, B. bassiana is a potential organism to be used in biological control of aphids. In a bioassay performed with doses of 1 x 10⁴- 1 x 10⁸ conidia mL^-1, the fungus showed a high level of virulence on the aphid Phorodon humuli, obtaining a LD50 of 1.37 x 10⁵ conidia mL^-1 and TL50 reduced as conidia dose increased (^{Dorschner et al., 1991}). Something similar happened in this paper because higher doses had the lowest values in TL50 (Table 6).

Whereas cited, B. bassiana is a fungus that can be used for biological control of populations of L. zonatus in an innocuous way, since it does not generate pollution to the environment; however, there is the potential risk of damaging some beneficial organisms. Because of this, there is need to perform studies in greenhouse and field to validate the effectiveness of this fungi against L. zonatus under different conditions.