Diaphorina citri Kuwayama (Hemiptera: Liviidae) is one of the most important invasive pests in the citrus industry of the world; this qualification has been assigned mainly by its role as a vector of Candidatus Liberibacter, a bacterium associated with huanglongbing (Bové 2006; Yang et al. 2006). Nowadays, huanglongbing is considered as one of the most devastating diseases that plague the crop worldwide; its management has been achieved basically through the elimination of infected plants, use of certified plant material, and vector control (Bové 2006; Gottwald et al. 2007; National Research Council 2010; da Graça et al. 2015). Presently, effective eradication methods against the rapidly dispersing vector are unavailable (Bové 2006). Management of D. citri has been constrained to the use of chemical control (Yang et al. 2006; Belasque et al. 2010; Stansly et al. 2013; Qureshi et al. 2014). However, intensive use of pesticides has began to produce collateral effects that are noticed in resistance selection to insecticides in the vector (Yang et al. 2006; Tiwari et al. 2011), resurgence of controlled key pests, and outbreaks of secondary pests (Yang et al. 2006; Monzó et al. 2012).

Vector management needs to be addressed toward the integration of methods that allow a reduction in the use of insecticides as well as in insect population density and huanglongbing infection and dispersion rate (Yang et al. 2006; Stansly et al. 2013). Some beneficial insects (Aubert 1990; Hoy & Nguyen 2001; Michaud 2004; López-Arroyo et al. 2009; Qureshi & Stansly 2009; Cortez-Mondaca et al. 2010) and entomopathogens have been considered as biological control agents of D. citri (Subandiyah et al. 2000; Yang et al. 2006; Meyer et al. 2007; Avery et al. 2009). Easy integration of fungi with pesticides has motivated the exploration of diverse fungal species, e.g., Beauveria bassiana (Bals.-Criv) Vuill. (Hypocreales: Cordycipitaceae) (Padulla & Alves 2009; Gandarilla-Pacheco et al. 2013; Casique-Valdes et al. 2015), Hirsutella citriformis Speare (Hypocreales: Ophiocordycipitaceae) (Subandiyah et al. 2000; Meyer et al. 2007; Casique-Valdes et al. 2011; Hall et al. 2012; Romero-Rangel et al. 2012; Cortez-Madrigal et al. 2014; Orduño-Cruz et al. 2015; Perez-Gonzalez 2015a,b), Isaria fumosorosea Wize (= Paecilomyces fumosoroseus) (Hypocreales: Cordycipitaceae) (Subandiyah et al. 2000; Meyer et al. 2007; Hoy et al. 2010; Gandarilla-Pacheco et al. 2013), and Lecanicillium lecanii (Zimm.) Zare & W. Gams (= Verticillium lecanii) (Hypocreales: Cordycipitaceae) (Rivero-Aragon & Grillo-Ravelo 2000).

Hirsutella citriformis was found producing remarkable natural epizootics on D. citri in the field (Aubert 1987; Subandiyah et al. 2000; López- Arroyo et al. 2009). Due to its characteristic sporulation in the insect (Hall et al. 2012), visual detection of attacked specimens in the trees frequently suggest a potential high value of the fungus for D. citri biological control. However, Hall et al. (2012) indicated an estimated contribution of H. citriformis in the reduction of vector populations by 4%. Hence, it is necessary to expand the exploration for highly pathogenic H. citriformis strains that allows re-evaluating the role that this fungus species may have for biological control of D. citri. Variability in pathogenicity among strains of H. citriformis may occur (Orduño-Cruz et al. 2015; Pérez-González et al. 2015a), as it was reported for other species of entomopathogenic fungi, even in those tested against D. citri (Mena et al. 2003; Hoy et al 2010). Thus, this factor may allow identifying more aggressive strains with the potential to cause increased mortality in D. citri while it proves to be harmless to other non-target insects.

Table 1.

Hirsutella citriformis monoconidial strains isolated from Diaphorina citri obtained from 8 citrus-growing areas of Mexico.

The objective of this study was to evaluate 8 native strains of H. citriformis, obtained from diverse areas of the Mexican citriculture, against D. citri adults, and to test non-target effects in the predators Chrysoperla rufilabris (Burmeister) (Neuroptera: Chrysopidae) and Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae), beneficial insects that attack D. citri in areas where the fungus has been found in Mexico (Cortez-Mondaca et al. 2010).

Materials and Methods

Results

VIRULENCE AGAINST D. CITRI

All H. citriformis strains were able to kill D. citri adults in the contact bioassays (Fig. 1; Table 2). Mortality was observed beginning 6 d post exposure to conidia in a range from 1.8 to 8.2%, with the highest mortality in insects exposed to strain INIFAP-Hir-1 and the lowest in untreated controls (Fig. 1). At 10 d after inoculation, we observed occurrence of the characteristic H. citriformis synnemata on D. citri cadavers. At 13 d after application, the evaluated fungal strains caused 25.8% (INIFAP-Hir-6) to 49.9 % mortality (INIFAP-Hir 1) in D. citri (Fig. 1). This trend of mortality continued among the treatments until the last record date, which was 26 d after exposure (Fig. 1), when overall mean mortality caused by strain INIFAP-Hir-1 in the 3 replicated bioassays ranged from 88.1 to 98.3%, followed by IB-Hir-2 (87.4–89.3%), whereas mean mortality in the untreated controls ranged from 7.1 to 18.5% (Fig. 1; Table 2). In the 3 bioassays, there were significant differences in overall mortality among treatments (bioassay 1: F = 19.31; df = 8,45; P < 0.001; bioassay 2: F = 32.36; df = 8,45; P < 0.001; bioassay 3: F = 44.12; df = 8,45; P < 0.001). Observed mycosis by H. citriformis ranged from 48.7% (INIFAP-Hir-5) to 81.2% (IB-Hir-2) (Table 2); dead specimens in the control did not show any sign of mycosis (Table 2).

Fig. 1.

Overall mean mortality of Diaphorina citri adults caused by conidia of 8 Hirsutella citriformis strains applied by contact (3 separate bioassays) under controlled conditions (26 ± 1 °C, 76 ± 4% RH, 16:8 h L:D photoperiod). Error bars represent the standard error (n = 7).

Table 2.

Mortality of Diaphorina citri caused by conidia of 8 Hirsutella citriformis strains applied by contact (3 separate bioassays) under controlled conditions (26 ± 1 °C, 76 ± 4% RH, 16:8 h L:D photoperiod).

When conidia were applied by spraying, INIFAP-Hir-1 and IB-Hir-2 strains caused 69% overall mortality in D. citri after 26 d; this value was significantly higher than that recorded in the untreated control (F = 23.48; df = 2,18; P < 0.001; Tukey's test, α = 0.05) (Fig. 2). In this test, we observed H. citriformis mycosis in dead specimens ranging from 20.5 to 62.7%.

In the test of H. citriformis blastospores in spray inoculation against D. citri, initial mortality was observed 6 d after the fungus application and ranged from 6.2% (INIFAP-Hir-2) to 13.9% (INIFAP-Hir-1), whereas mortality in the untreated control was 4.1% (Fig. 3). At 12 d post application, H. citriformis blastospores caused 20.4 to 38.0% mortality of D. citri (Fig. 3). After 26 d, the INIFAP-Hir-2 and INIFAP-Hir-5 strains sprayed as blastospores produced the highest overall mortality (48.7%) in D. citri; this value was statistically different from that recorded in the control (17.6%) (F = 4.68; df = 5,36; P = 0.002; Tukey's test, α = 0.05) (Fig. 3). Occurrence in humid chambers of mycosis by H. citriformis in D. citri dead specimens from the treatments with blastospore application was very low, approximately 10%; in the case of cadavers from the control, mycosis was null. Probit analysis showed that LC50 for strain INIFAP-Hir-1 was 3.40 × 10⁶ conidia per mL, with 95% confidence interval 1.38 to 8.51 × 10⁶ conidia per mL. LC90 was 2.67 × 10⁹ conidia per mL, with 95% confidence interval 2.18 × 10⁸ to 2.44 x 10¹¹ conidia per mL. The regression equation obtained was y = 4.31 + 0.44x (χ2 = 7.0055; P = 0.05).

Fig. 2.

Mean mortality of Diaphorina citri caused by conidia sprays of 2 Hirsutella citriformis strains under controlled conditions (26 ± 1 °C, 76 ± 4% RH, 16:8 h L:D photoperiod). Different letters indicate significant differences (Tukey's test, α = 0.05). Error bars represent the standard error (n = 7).

PATHOGENICITY AGAINST PREDATORS

Overall mortality by H. citriformis in H. convergens adults ranged from 9% (INIFAP-Hir-1) to 11% (INIFAP-Hir-2) after 24 d of conidia application; these percentages did not differ significantly from that recorded in the untreated control (F = 0.1623; df = 4,20; P = 0.953) (Table 3). Dead specimens did not develop mycosis after being placed in a humidity chamber. In C. rufilabris, overall mortality by H. citriformis 30 d after application in larvae and recorded in emerged adults ranged from 19% (INIFAP-Hir-1) to 25% (INIFAP-Hir-2); there were no statistical differences among treatments (F = 0.577; df = 4,15; P = 0.684) (Table 3). As in H. convergens, C. rufilabris dead specimens did not develop mycosis by H. citriformis.

Fig. 3.

Mean mortality of Diaphorina citri caused by blastospores of 5 Hirsutella citriformis strains under controlled conditions (26 ± 1 °C, 76 ± 4% RH, 16:8 h L:D photoperiod) during 26 d post inoculation. Different letters indicate significant differences (Tukey's test, α = 0.05). Error bars represent the standard error (n = 7).

Table 3.

Overall mean mortality of the predators Chrysoperla rufilabris and Hippodamia convergens at 30 d and 24 d, respectively, after conidia of Hirsutella citriformis strains were applied by contact.

Discussion

In our study, all the tested Mexican H. citriformis strains showed potential as biological control agents of D. citri as they performed well in the bioassays using inoculation by conidia contact, by which they caused high mortality rates that were close to 90% (Table 2). Similar to the results of Meyer et al. (2007), we observed that mortality of D. citri specimens began 6 d after exposure to the fungus (Fig. 1); occurrence of characteristic H. citriformis synnemata on D. citri cadavers was observed 10 d after inoculation, which agreed with results by Casique- Valdés et al. (2011); however, the highest accumulated mortality (90%; Fig. 1; Table 2) in our study was recorded until 26 d after the application of the fungal strains, whereas Casique-Valdés et al. (2011) and Orduño-Cruz et al. (2015) reported 100% mortality just 6 d after inoculation, and Meyer et al. (2007) obtained the same rate at 9 d post inoculation. These differences in achieving the highest mortality could be due to genetic variability of the evaluated strains that could be associated with high pathogenicity as observed in many pathogens (see Mena et al. 2003; Hoy et al. 2010); hence, we were expecting some strain variability.

In addition, variations in bioassay procedures such as the use of different fungal conidia doses could explain the above differences; in Meyer et al. (2007) and Casique-Valdés et al.(2011), doses were unreported, and we realized based on the size of conidia (see Speare 1920; Perez-Gonzalez et al. 2015b) that Orduño-Cruz et al. (2015) could have overestimated the number of conidia attached to the insect and consequently used a very high dose of conidia, probably far beyond to the LC90 of 2.6 × 10⁹ conidia per mL estimated in our bioassay with sprayed conidia. Meyer et al. (2007) and Casique-Valdés et al. (2011) might have used a dose in these amounts. It is also possible that the first dead specimens observed in our trial were those that were infected with a greater number of conidia and that those that died later barely had received a lethal dose. Such differences in the number of conidia per insect could be due mainly to the inoculation method by not providing a homogeneous presence of conidia on the insect body.

Nonetheless, we emphasize that the observed mortality curves (Fig. 1) may resemble a possible pattern for infected D. citri by H. citriformis in nature. Because the H. citriformis conidia are characteristically sticky (Speare 1920; Meyer et al. 2007; Perez-Gonzalez et al. 2015b), they would not spread readily as seen in other entomopathogenic fungal genera such as Beauveria or Metarhizium (Mena et al. 2003; Padulla & Alves 2009). Thus, it is possible that H. citriformis would exhibit a behavioral manipulation of the host as it has been documented for Ophiocordyceps unilateralis (Tul. & C. Tul.) Petch (Hypocreales: Ophiocordycipitaceae) in ants (Andersen et al. 2009; Pontoppidan et al. 2009). Hirsutella citriformis could use of the host as a vehicle for dispersion. For instance, numerous healthy hosts could be infected with a lethal, sublethal, or innocuous concentration of conidia that eventually will kill the insect through time and on several host plants, possibly far away from the initial site of infection. This scenario would reflect a possible strategy or adaptation of the fungus to spread and survive as suggested by Casique-Valdés et al. (2011) and Hall et al. (2012).

Research on H. citriformis has been neglected, and there are many uncovered areas of study. From the stated above, an essential need is to demonstrate the mechanisms involved in the possible host manipulation, as well as to understand completely its role at the insect population level and, therefore, the settings and development of epidemics.

In the bioassays in which conidia were applied by spraying, mortality caused by the 8 strains was remarkably reduced (Fig. 2). Such low values when conidia in suspension were used for inoculation was also observed by Orduño-Cruz et al. (2015) in H. citriformis. A possible explanation for these results could be the damage or removal of the water-soluble mucilaginous envelope of conidia during the preparation of inoculum (Sánchez-Peña et al. 2011). These changes eventually could result in poor adherence to the cuticle of the host, as well as in low germination, decreased virulence, and consequently reduced host mortality as the one that we obtained in our tests (Fig. 2). We used acacia gum to overcome such factor; however, it was ineffective and it has prompted the need for further research in application techniques of the fungus that could support its successful use in the field.

The LC50 and LC90 determined for the strain INIFAP-Hir-1 in our study (3.4 × 10⁶ and 2.6 × 10⁹ conidia per mL, respectively), applied by spraying of conidia, are the first reported for H. citriformis. Hoy et al. (2010) determined for I. fumosorosea against D. citri LC50 and LC95 values of 6.8 × 10⁵ and 2.2 × 10⁸ conidia per mL, respectively. Although the LC values are relatively similar for both fungal species, H. citriformis due to its slow development would be more expensive to use in an extensive way. For example, the conidia of the H. citriformis strains used herein germinated after 48 to 72 h (O. P. G., unpublished data) and growth is reportedly slow (radial growth = 0.083 to 0.114 cm/d) (Meyer et al. 2007; Pérez-González et al. 2015a). For I. fumosorosea, Vu et al. (2009) indicated conidia germination within 12 to 24 h, and according to Cabanillas & Jones (2009), this fungus showed fast radial growth (0.33 cm/d); both traits were at least 3 times greater than in H. citriformis. With the estimated LC50 and LC90 for H. citriformis against D. citri as reference, its potential use for biological control of this insect requires investigation to facilitate the commercial production of the fungus and make it economically viable.

In the evaluation of 5 H. citriformis Mexican strains as blastospores, we found that they induced low mortality against D. citri. Similar results were obtained previously by Pérez-González et al. (2015a). In our bioassays, the average mortality ranged from 31 to 48% (Fig. 3), and mycosis occurred only in a small number of insects. A higher mortality in D. citri with the use of blastospores was reported by Orduño-Cruz et al. (2015); they observed 61 to 65% mortality caused by 2 strains of H. citriformis isolated from unidentified species of insects; mycosis data were unavailable. Low values of mortality and mycosis produced by blastospores recorded in this study may have been caused in part by toxins released during H. citriformis liquid-culture growth, as reported by Liu et al. (1995) and Rosas-Acevedo et al. (2003) for strains of Hirsutella thompsonii F. E. Fisher. Our mortality data suggest that the application of conidia for control of D. citri is preferable over the use of blastospores; however, it is important to continue determining conditions under which this fungus develops infective and virulent blastospores against D. citri as their use could be more practical and they could be produced in high numbers in liquid culture in a short time (Avery et al. 2009; Romero-Rangel et al. 2012).

Tests of H. citriformis conidia inoculation by contact against H. convergens adults and C. rufilabris larvae produced low mortality without significant differences among treatments, including the untreated controls (Table 3). Data concerning H. convergens adults were similar to those reported by Gandarilla-Pacheco et al. (2012) in tests with B. bassiana and I. fumosorosea, in which they recorded 6 to 25% mortality. In contrast, the effect on C. rufilabris (18 to 25% mortality; Table 3) was lower than that reported by these authors for Ceraeochrysa valida (Banks) and Eremochrysa punctinervis (McLachlan) (Neuroptera: Chrysopidae) larvae, for which they recorded 50% mortality caused by a local strain of I. fumosorosea (Gandarilla-Pacheco et al. 2012).

Our study confirms the value of including tests for non-target effects. We here evaluated 4 H. citriformis strains under a hypothetical scenario of causing high mortality in diverse organisms different to our target. Hence, we would have the opportunity to select a possible fungal strain that produces no harmful effects for other organisms, thereby evading antagonistic interactions (Thomas & Lynch 2003) and allowing a relatively safe use for our evaluated agent for biological control. As there are many species of arthropods associated with citrus (Monzó et al. 2012), and as we tested the fungus in only 2 beneficial entomophagous insect species, it would be imprecise to state that H. citriformis appears to be a specific natural enemy for D. citri. Instead, we suggest further studies of this valuable natural enemy aimed to generate in the short term knowledge for its successful and safe use as a biological control agent. Plans should be made for its integration with other beneficial arthropods for a sustainable management of D. citri (Thomas & Lynch 2003), generating in this way alternatives to the present and intense use of pesticides against the vector of huanglongbing.

We conclude that the 8 Mexican fungal strains of H. citriformis tested against D. citri were pathogenic to the Asian citrus psyllid adults, with variability in performance associated with strain diversity. Among the strains, INIFAP-Hir-1 showed constantly favorable results. At the present time, use of conidia seems to be the best method for application of this fungus; the use of blastospores produced in liquid culture requires further investigation to determine optimal conditions to develop infective and virulent propagules against D. citri. Our data obtained from tests against the predators C. rufilabris and H. convergens could support the safe use of H. citriformis as a biological control agent of D. citri.