Endophytic potential of entomopathogenic fungi associated with Urochloa ruziziensis (Poaceae) for spittlebug (Hemiptera: Cercopidae) control

2.1 Origin of entomopathogenic fungi and seeds of U.  r uziziensis

Fungi isolated in a silvopastoral system in the state of Maranhão, Brazil, Fusarium sp. (Hypocreales: Nectriaceae; UFMG 11443, GenBank = ON831395) and Metarhizium anisopliae (Metschn.) Sorokīn (Hypocreales: Clavicipitaceae; UFMG 11444, GenBank = ON831396) were provided by the entomopathogens collection of the Department of Biosystems Engineering (DEPEB), UFSJ, maintained since 2017. These fungi are deposited in the Microorganism Collection of the Federal University of Minas Gerais, Brazil (World Data Center for Microorganisms - WDCM 1029. CM-UFMG). The DNA sequences were analyzed and compared with the type culture sequences deposited in GenBank using the BLASTn program (Basic Local Alignment Search Tool version 2.215 of BLAST 2.0) available from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/blast/). The other strain of M. anisopliae, strain E9 (1.39 × 10⁸ conidia/g), was purchased commercially from KOPPERT do Brasil Holding S.A^® (Piracicaba, São Paulo, Brazil). The U. ruziziensis cv. Kennedy seeds were purchased commercially from SOESP Company (Presidente Prudente, São Paulo, Brazil).

2.2 Fungal inoculation of seeds

For each fungus, 100 g of seeds was used. After weighing the seeds, superficial disinfestation was performed by washing the seeds in sodium hypochlorite (2 %) for 2 min and then in 70 % alcohol for 1 min. After these steps, the seeds were rinsed with distilled water according to the methodology adapted from Carvalho et al. (2012) and Ferreira et al. (2017). Batches of conidia were tested for their viability according to the methodology proposed by Lopes et al. (2013). For fungal inoculation, 400 mL of sterile distilled water suspension containing 1 × 10⁸ conidia/mL was added, as specified for the commercially acquired M. anisopliae strain, and 0.05 % Tween 80 was included as a surfactant. A control suspension without the presence of fungi was prepared, for a total of four treatments. The seeds were in contact with the suspension for 30 min according to an adaptation of the methodology by Keyser et al. (2014).

2.3 Cultivation of U. ruziziensis containing entomopathogenic fungi

Ten U. ruziziensis seeds treated with entomopathogenic fungi were deposited on a soil, sand, and manure mixture at a ratio of 3:1:1 (Resende et al. 2012) and were covered with a layer of vermiculite (Micron^® Jacareí, São Paulo, Brazil) in a 2L planter. Every 45 days the soil was fertilized with urea (0.09 g/planter), simple superphosphate (0.04 g/planter), and potassium chloride (0.04 g/planter). The plants were irrigated by MA-50 micro-sprinklers with anti-drops (Photogenesis, Belo Horizonte, Minas Gerais, Brazil) with a capacity of 50 L/h/m², automatically, three times a day for 15 min throughout the experiment. After 45 days, the first pruning was performed, leaving the plants 15 cm tall.

2.4 Pest insect collection

In October 2022, adults of M. spectabilis and D. schach were collected from the experimental field of Embrapa Dairy Cattle in Coronel Pacheco, Minas Gerais State, Brazil with an entomological net. The captured adults were placed in entomological cages at the Entomology Laboratory at Embrapa Dairy Cattle. After five days, eggs of the two pest species were collected following the methodology of Auad et al. (2007). Then, 50 eggs of each species were placed on the U. ruziziensis, in the vegetative phase (40 cm high), without the presence of fungi, in the greenhouse and covered with voile fabric to prevent the nymphs from escaping after hatching. Forty planters were infested, totaling 2,000 eggs/species. After 35 days, the nymphs obtained from the planters were used in the experiments.

2.5 Molecular analysis of fungi

After macro- and micromorphological analysis, the fungal isolates were grouped and from each group a random sample was taken with characteristics similar to the fungi applied for molecular analysis, totaling: 12 samples for experiment 1; eight samples for experiment 2, 3, and 4. Filamentous fungi were inoculated on dextrose (4 %), casein (1 %) and agar (1.5 %; Sabouraund dextrose agar, KASVI^®, Spain) for seven days. The DNA of the isolates was extracted from the microorganism grown in culture medium according to the method described by Doyle and Doyle (1987). The extracted genomic DNA sample was submitted to the polymerase chain reaction (PCR) to amplify the internal transcribed spacer (ITS) region of the rDNA using the SR6R (5′ – AAGWAAAAGTCGTAACAAGG – 3′) and LR1 (5′ – GGTTGGTTTCTTTTCCT – 3′) primer oligonucleotides (Vilgalys and Hester 1990). The PCR mixture consisted of 1 µL of DNA, 1 µL of each primer at 10 µM, 10 µL of 5X PCR buffer, 1 µL of dNTPs at 10 mM, 0.2 µL of GoTaq DNA polymerase 5U/µL (Promega), and 35.8 µL autoclaved MilliQ H₂O, for a final volume of 50 µL. The amplification program consisted of initial denaturation at 94 °C for 2 min followed by 40 cycles of denaturation at 94 °C for 10 s, annealing at 54 °C for 30 s, extension at 72 °C for 45 s, and final extension at 72 °C for 4 min. The amplified products were verified by electrophoresis on a 0.8 % agarose gel stained with ethidium bromide. The amplified products were purified by precipitation with polyethylene glycol (Schmitz and Riesner 2006), submitted to the sequencing reaction by the chain termination method using the Big Dye 3.1 reagent (Applied Biosystems) and analyzed in a 3,500 xL automatic capillary sequencer (Applied Biosystems).

2.6 Experiment 1: evaluation of the frequency of infection and persistence of fungi in plant tissues

U. ruziziensis seeds were treated with the three fungal strains and the control solution without the presence of fungi. The seeds were deposited on the soil, sand and manure mixture at a ratio of 3:1:1 (Resende et al. 2012), and were covered with a layer of vermiculite (Micron^®, Jacareí, São Paulo, Brazil), in a 2 L planter, and kept in a greenhouse.

Plant tissue samples (top leaf) were taken at random points from plants in 15 planters, totaling 60 leaf samples (one sample × four treatments × 15 pots/replication) at 45, 120, and 150 days after sowing. These leaves were put into sterile plastic tubes, placed in a thermal box and transported to DEPEB (UFSJ) for testing. Each leaf was surface disinfected by immersion in 70 % ethanol (1 min) and 2 % sodium hypochlorite (1 min), followed by washing with sterile distilled water (2 min) (Carvalho et al. 2012; Ferreira et al. 2017). After disinfection, a fragment of each leaf was placed in a Petri dish containing Sabouraud dextrose agar culture medium (4 % dextrose, 1 % casein, and 1.5 % agar; Kasvi^®). The plates were incubated at 25 °C ± 2 °C for approximately 7 days in an EletroLab^® BOD-type climatized chamber. To verify the presence of the inoculated fungi, the macromorphology and micromorphology of the fungi present in the fragments were compared to the three inoculated fungal strains according to methodology adapted from Fróhlich et al. (2000). The fungal species were grouped using identification keys according to Alves (1998) for subsequent molecular analysis (see section above).

The experimental design used was completely randomized with plants from seeds treated with different fungi or left untreated (control). The treatments were represented by the leaves collected randomly from each pot for the analysis of the frequency of infection by the fungi applied. Endophytic confirmation was adopted when more than 60 % of the samples presented the characteristics of the fungi applied.

2.7 Experiment 2: efficacy of entomopathogenic fungi applied via seed and confirmation of infection, for the control of M. spectabilis and D. schach in a greenhouse

The experimental design was completely randomized in a factorial scheme (4 × 2) consisting of seeds treated with the three fungal strains or the control solution and two species of spittlebugs, M. spectabilis or D. schach, with 10 replications per treatment, totaling 80 planters.

Ninety days after sowing, 10 nymphs (from the third to fifth instar) of each species were placed in each planter. Daily, for a period of 10 days, the nymphs that died and the adults that emerged and died were removed, packed in 1.5 mL microcentrifuge tubes and labeled according to treatment. After this period, on the 10th day, dead and alive nymphs and adults were collected for analysis of the presence of the fungi. Ten adults coming from nymphs that did not die under the action of the fungi were placed on the aerial part of the same plant from each treatment. Ten days after placing for a second time, the insects that were dead were removed (after 20 days of exposure for that individual), packed in 1.5 mL microcentrifuge tubes and labeled according to treatment. The mortality data (%) in the different treatments were used to calculate the efficiency of the fungi in the greenhouse bioassay.

Samples of nymphs and/or adults of spittlebugs were collected into sterile 1.5 mL microcentrifuge tubes to analyze the cause of mortality and placed in thermal boxes. The samples were taken to the DEPEB at UFSJ for incubation in the middle of culture medium selective for fungi (Sabouraud dextrose agar, 4 % dextrose, 1 % casein, and 1.5 % agar; Kasvi^®) to evaluate the presence of fungi in the nymphs and adults of spittlebugs, which were applied in the treatment of U. ruziziensis seeds. Superficial disinfestation of insects, fungal isolation and identification were performed as described for Experiment 1.

2.8 Experiment 3: efficacy of entomopathogenic fungi applied via seeds and used on banker plants to control M. spectabilis under field conditions

Ten U. ruziziensis seeds treated with the three fungal strains and seeds from the control group, without any fungus, were planted in the greenhouse, as described for Experiment 1. When the plants had been sown for 100 days, 10 pots from each treatment plus the control group were taken to the experimental field and placed in clumps of elephant grass (Pennisetum purpureum Schumach.; Poaceae) with proven attack by M. spectabilis (to prove attack, screening and a visual search were carried out on the foam containing nymphs at the base of the plants). The pots were placed in a straight line with a distance of 5 m between them.

Every 7 days for a period of 3 weeks, the numbers of dead spittlebug adults from nymphs fed on elephant grass and that, upon emerging as adults, migrated to plants that were in planters, were determined. This count was performed in a radius of 2 m around each planter. Five samples were collected from each replicate per treatment, which were packaged and sent for analysis of the presence of the fungi in the insect tissues, as described in Experiment 1.

2.9 Experiment 4: analysis of the persistence of fungi in tissues of U.  ruziziensis and infection of M. spectabilis on plants of different ages

After treatment with the three fungi strains or no fungal treatment, the U. ruziziensis seeds were stored for 60 days in a climatized chamber (22 °C). From this moment on, seed samples were collected to germinate every 30 days for 10 consecutive months. The plants obtained on each of the sowing dates were kept in 2 L planters in a greenhouse with controlled fertilization, irrigation and pruning to maintain an approximate height of 25 cm. The aerial part of the plants of batch from one to nine were pruned at 45 days, leaving the plants 15 cm tall. Plants in the last batch of seeds (the youngest plants) were 45 days old and so were not pruned. So, when infested with M. spectabilis nymphs the aerial part of all plants was 45 days-old.

A randomized block design was used with plants from seeds treated with different fungi strains or untreated (control) with plants of different ages (1–10 months), with five replications. When the plants of the last batch of seeds were 45 days old, the plants of all 10 batches (plant age from 45 days to 10 months, sowing times) were infested with four nymphs of the third and fourth instars of M. spectabilis per pot to evaluate the presence of the fungus and its efficiency in controlling the insect and to define the persistence of the fungus in stored seeds and in vivo plants managed with elimination of the aerial part (cuts) to simulate natural conditions in a pasture.

For analysis of the frequency of fungal infection in plant tissue (leaves), a random sample was taken from each plant, totaling five samples per treatment, placed in a sterile 1.5 mL microcentrifuge tube, placed in a thermal box and taken to the Microbiology Laboratory of the Federal University of São João Del Rei for testing.

For analysis of the frequency of fungal infection in the pest insects fed on the plants from the different sowing lots, four nymphs of M. spectabilis were allocated per planter, and to prevent the nymphs of the insect pest from escaping, the planters were covered with voile. After 10 days of insect feeding in different treatments, dead and live nymphs were removed and placed in 1.5 mL microcentrifuge tubes. They were then frozen at −22 °C and sent to DEPEB, UFSJ, Minas Gerais, Brazil, to verify the presence of fungi in insect tissues. Superficial disinfestation of insects and leaves of U. ruziziensis and fungal isolation and identification were performed as described for Experiment 1.

2.10 Statistical analyses

All data analyses were performed with R version 4.2.2 (R Core Team 2022). The data obtained did not meet normality assumptions related to the residuals and homogeneity of variances (Shapiro–Wilk test and Bartlett test, p < 0.01). A generalized linear model (GLM) followed by analysis of variance (ANOVA) was used to verify whether there were significant differences in the proportions of plants and spittlebugs expressing the presence of endophytic fungi and the mortality of spittlebugs that fed on plants inoculated with the endophytic fungi. For these comparisons, a GLM was used in a logistic regression model with a binomial distribution, and the “hnp” package (Moral et al. 2017) was previously used to choose the best model. In this model, the effects of treatments (fungal species, spittlebug species, developmental stage, and storage time) were tested individually, as were their interactions. In instances where the impact of interactions did not attain statistical significance, the effects of treatments were assessed independently. The treatments were separated using logistic regression, which allows the prediction of values taken by a categorical variable, normally binary, from a series of continuous and/or binary explanatory variables. This is a regression model for binomially distributed dependent variables. Thus, it is a generalized linear model that uses the logit function as the link function. This model does not assume normality of residuals or homogeneity of variances. The presence or absence of the endophytic fungi in the plants or spittlebugs was included as a variable in the model. The packages visreg (Breheny and Burchett 2019), MASS (Ripley 2019), ggplot2 (Wickham 2016), and GGally (Schloerke et al. 2021) were used for the logistic regression.

3.1 Frequency of infection and persistence of fungi in plant tissues

The interactions (fungal species and period of permanence of fungi in plants) did not attain statistical significance; hence, the effects of treatments were assessed independently. The entomopathogenic fungi Fusarium sp. (UFMG 11443), M. anisopliae (UFMG 11444), and M. anisopliae (commercial strain) were isolated from the tissues of U. ruziziensis 45 days after seed treatment, which confirmed their endophytic capacity. The presence of the fungi used in the treatments was confirmed by molecular analysis (Table 1). The percentage of plants with the fungi in their tissues was above 60 %, which was significantly higher (χ² = 399.72; df = 3; P < 0.0001) than the percentage of plants with fungi in their tissues obtained from untreated seeds (Figure 1A). This percentage did not differ significantly among the plants obtained from the seeds that were treated with different fungi. In addition, it was found that the fungi in plants was reduced significantly 150 days after sowing compared with 45 days after sowing (χ² = 386.12; df = 2; P = 0.0011) (Figure 1B).

Figure 1:

Entomopathogenic fungi Fusarium sp. (UFMG 11443, GenBank = ON831395), Metarhizium anisopliae (UFMG 11444, GenBank = ON831396), and Metarhizium anisopliae (commercial strain) present on the leaves of Urochloa ruziziensis at 45 days of age (A). Presence of entomopathogenic fungi in plants of different ages (B). Significant differences are indicated by different letters above bars.

3.2 Isolation of fungi from spittlebugs and insect pest mortality in a greenhouse

The interactions (spittlebug species and insect developmental stage) did not attain statistical significance; hence, the effects of treatments were assessed independently. The presence of the entomopathogenic fungi isolated from nymphs was confirmed by molecular analysis (Table 1). The entomopathogenic fungi Fusarium sp. (UFMG 11443), M. anisopliae (UFMG 11444), and M. anisopliae (commercial strain) were isolated for up to and including 10 days from live nymphs and adults of M. spectabilis and D. schach fed on plants derived from seeds treated with the respective fungi. The presence of fungi did not differ significantly (χ² = 162.16; df = 1; P = 0.1839) between M. spectabilis and D. schach (Figure 2A) or between the developmental stages (χ² = 162.05; df = 1; P = 0.7419) of these insects (Figure 2B), demonstrating that the samples of the entomopathogenic fungi used affected both nymphs and adults of the spittlebug species. However, despite the confirmation of the presence of the fungi on the spittlebugs, the nymphal mortalities were below 20 % and did not differ significantly (χ² = 17.24; df = 3; P = 0.9853) from those of spittlebugs fed on plants from seeds not treated with the fungi (control) (Figure 2C).

Figure 2:

Infection and mortality of spittlebugs fed on plants grown from seeds treated with the entomopathogenic fungi Fusarium sp. (UFMG 11443, GenBank = ON831395), Metarhizium anisopliae (UFMG 11444, GenBank = ON831396), and Metarhizium anisopliae (commercial strain) in the greenhouse. Infection frequency of Mahanarva spectabilis and Deois schach (A). Adults and nymphs with the presence of entomopathogenic fungi (B). Mortality of spittlebug nymphs and adults in 10 days due to entomopathogenic fungi (C). Adult mortality (%) coming from nymphs that did not die under the action of the fungi in each treatment within 10 days, 10 days after placing the insects back on the aerial part of the plant (20 days of total exposure for that individual; D). Significant differences are indicated by different letters above bars.

Adults that died within 10 days, coming from nymphs that did not die under the action of the fungi within 10 days (20 days of exposure for that individual) were confirmed as infected by the endophytic fungi, with no significant differences in infection among the fungi applied. Only the control differed statistically from the other treatments, where the insects were not infected by the fungi used in the experiments (χ² = 39.14; df = 3; P = 0.0010) (Figure 2D).

3.3 Fungal isolation from spittlebugs and pest insect mortality under field conditions

The introduction of planters of banker plants from seeds treated with the fungi Fusarium sp. (UFMG 11443), M. anisopliae (UFMG 11444), and M. anisopliae (commercial strain) caused infection in M. spectabilis adults reared in elephant grass clumps in the field. In the M. spectabilis adults that fed on these plants and were collected dead nearby or on the plants, mortality was above 75 % for the three fungi, with infection confirmed by molecular analysis (Table 1), showing a significant difference (χ² = 48.27; df = 3; P < 0.0001) from the control treatment (Figure 3).

Figure 3:

Dead adults of Mahanarva spectabilis collected in the field around banker plants grown from seeds treated with entomopathogenic fungi Fusarium sp. (UFMG 11443, GenBank = ON831395), Metarhizium anisopliae (UFMG 11444, GenBank = ON831396), and Metarhizium anisopliae (commercial strain). Insects confirmed to be infected with entomopathogenic fungi. Significant differences are indicated by different letters above bars.

3.4 Analysis of the persistence of fungi in the tissues of U.  ruziziensis and infection of M. spectabilis on plants of different ages

There was no significant interaction (χ² = 255.11; df = 27; P = 0.9235) between the species of entomopathogenic fungi and the storage period of treated seeds/plant ages. Therefore, these factors were analyzed separately. The frequency of infection by entomopathogenic and endophytic fungi ranged from 5 % to 40 % in plants derived from seeds stored for 90 days after inoculation of the fungi (plants for 330 days) or stored for 360 days after inoculation (plants for 60 days); however, it was not significantly different (χ² = 295.88; df = 9; P = 0.1692) between plants measured on different days after inoculation. This evidence indicates the persistence of these fungi in plants and stored seeds, which was confirmed by molecular analysis (Table 1) of the fungi isolated from the plants over time (Table 2).

Insects fed on plants derived from seeds stored for 90 days (plants for 330 days) or stored for 360 days (plants for 60 days) showed a frequency of infection by entomopathogenic fungi from 21.8 % and 58.3 %, respectively; however, they did not present significant differences (χ² = 202.04; df = 9; P = 0.4636) for the time since fungal inoculation, which verified the persistence of fungi in seeds stored for up to 300 days and in plants 300 days after sowing (Table 2). The frequencies of fungal infection in plant tissues and M. spectabilis nymphs were significantly higher than that in the control treatment and did not differ from each other, with a mean of 28.6 % in plant tissues (χ² = 179.74; df = 3; P < 0.0001) and 53.8 % in insect pests (χ² = 48.27; df = 3; P < 0.0001) (Table 2).