Biological Control 90 (2015) 1–5
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Biological Control
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In vivo selection of entomopathogenic fungal isolates for control of Diaphorina citri (Hemiptera: Liviidae) ⇑ N. Orduño-Cruz a, A.W. Guzmán-Franco a, , E. Rodríguez-Leyva a, R. Alatorre-Rosas a, H. González-Hernández a, G. Mora-Aguilera b a Entomología y Acarología, Postgrado en Fitosanidad, Colegio de Postgraduados, Km. 36.5 Carretera México-Texcoco, Montecillo, Texcoco, Estado de Mexico 56230, Mexico b Fitopatología, Postgrado en Fitosanidad, Colegio de Postgraduados, Km. 36.5 Carretera México-Texcoco, Montecillo, Texcoco, Estado de Mexico 56230, Mexico highlights graphical abstract
Diaphorina citri is an important pest that transmits the disease HLB in citrus. In vivo selection of isolates was made based on mortality caused to D. citri. M. anisopliae and I. fumosorosea achieved 100% mortality with conidial suspensions. Hirsutella citriformis caused 100% mortality using dry conidia. Combination of inundative and inoculative biological control approaches are viable. article info abstract
Article history: The Asian citrus psyllid, Diaphorina citri, is an important pest of citrus, which transmits the disease Received 29 October 2014 Huanglongbing with devastating effects on the citrus industry around the world. Control relies mainly Accepted 19 May 2015 on synthetic pesticides, but biological control using entomopathogenic fungi may provide an alternative Available online 29 May 2015 strategy. Based on previous in vitro experiments, we selected four isolates, one from each of the species Beauveria bassiana, Metarhizium anisopliae, Isaria fumosorosea and Hirstella citriformis. With these isolates, Keywords: we carried out in vivo experiments to select an isolate based on its infection ability against adult D. citri. Asian citrus psyllid Using only H. citriformis isolates, we evaluated the mortality in D. citri achieved using blastospores. As a Hypocrealean fungi potential cause of the low mortality achieved with H. citriformis, we evaluated the effect of inoculum Hirsutella citriformis Isolate selection carrier (Tween 80 or distilled water) on conidial germination. We also compared the pathogenicity Conidial germination of dry conidia (not suspended in 0.03% Tween 80) of H. citriformis and M. anisopliae against adult D. citri. Using conidial suspensions, all isolates caused mortalities above 80% in adult D. citri cohorts, except H. citriformis, which caused a maximum of 40% mortality. The mortality caused by blastospore suspensions of H. citriformis, were 60%. Tween 80 solution and distilled water significantly reduced the germination of H. citriformis conidia. Dry conidia of H. citriformis and M. anisopliae caused 100% mortality. Our results suggest that using a combination of biological control approaches, both inundative (using M. anisopliae or I. fumosorosea isolates in suspension) and inoculative (using H. citriformis as dry conidia in autoinoculation devices), has good biological control potential. The suitability of the combined use of these two biological control approaches for management of D. citri is discussed. Ó 2015 Elsevier Inc. All rights reserved.
⇑ Corresponding author. Fax: +52 595 9520200x1607. E-mail addresses: [email protected], [email protected] (A.W. Guzmán-Franco). http://dx.doi.org/10.1016/j.biocontrol.2015.05.011 1049-9644/Ó 2015 Elsevier Inc. All rights reserved. 2 N. Orduño-Cruz et al. / Biological Control 90 (2015) 1–5
1. Introduction 2.2. Fungal isolates and production of inoculum
Huanglongbing disease (HLB), is currently the most lethal Four isolates, one from each of the species B. bassiana s.l., I. disease affecting citrus; in conjunction with its vector Diaphorina fumosorosea, M. anisopliae s.l. (Metschn.) Sorokı¯n (Anamorphic citri (Hemiptera: Liviidae), it represents a major threat to the citrus Clavicipitaceae) and H. citriformis were used (Table 1). These iso- industry (Grafton-Cardwell et al., 2013; Hall et al., 2013). Single lates were selected because they achieved the greatest vegetative control strategies are not sufficient and combined strategies growth at a range of temperatures, and had greater sporulation implemented at a regional scale are needed. For example, for HLB and germination than other isolates in in vitro experiments management, the use of certified plants and preventative removal (Orduño-Cruz et al., paper submitted). For experiments, all isolates of infected plants in combination with control of its vector are rec- were retrieved from long-term storage at À80 °C and grown on ommended when extensive areas are infested (between 500 and Sabouraud Dextrose Agar (SDA) plates at 25 °C in complete dark- 1000 ha) (Bové, 2006; SENASICA, 2012). Currently, the manage- ness for 15 days (40 days for H. citriformis) prior to experimenta- ment of HLB involves constant monitoring of the vector (D. citri) tion. Isolates were not subcultured more than three times after in combination with biological and chemical control methods; retrieval from À80 °C storage. For the blastospores experiment, the use of insecticides still represents the most widely used an additional H. citriformis (H1) isolate was included for compar- method for the control of D. citri in countries such as Brazil, USA ison (Table 1). and Mexico (Manjunath et al., 2010; Tiwari et al., 2011; After the appropriate incubation time conidial suspensions of SENASICA, 2012; Grafton-Cardwell et al., 2013). It is well know all isolates were prepared using the following method: conidia that using chemical insecticides as the main control strategy is were separated from SDA plates using a bacterial loop and sus- not sustainable, and has known negative side effects such as envi- pended in 5 mL of 0.03% Tween 80 in a 50 mL centrifuge tube. ronmental pollution, encouragement of secondary pests and reduc- Suspensions were vortexed for 2 min. and then filtered into a tion of natural enemy populations (Doutt and Smith, 1971; Qureshi new 50 mL centrifuge tube through a sterile cloth to remove any and Stansly, 2009; Tiwari et al., 2011). remaining mycelia. A 1:1000 dilution was made from each suspen- Natural infections of D. citri by entomopathogenic fungi in sion, the conidia concentration estimated using a haemocytometer Mexican field populations (NOC, personal observation) and else- and then adjusted accordingly to a final concentration of where (Avery et al., 2011; Casique-Valdes et al., 2011; Hall et al., 1 Â 108 conidia mLÀ1. 2013; Meyer et al., 2007), suggest these fungi have great potential as biological control agents of D. citri within integrated control strategies. During 2011 fungal isolates were collected from 2.3. Susceptibility of adult D. citri to infection by conidia of four field-infected adult D. citri and other unidentified individuals from selected isolates the family Cicadellidae in Mexico; the majority were isolates of Hirsutella citriformis Kuwayama (Anamorphic Ophiocordyceps), Replicate groups of 20, 5–8 days old, mixed-sex adult D. citri with some additional isolates of Isaria fumosorosea Wise and were used. Different groups of 20 adults were inoculated with 8 À1 Beauveria bassiana (Bals.-Criv.) Vuill. (Anamorphic 1 Â 10 conidia mL suspensions of each fungal isolate (H2, B1, Cordycipitaceae) (Orduño-Cruz et al., paper submitted). Sixteen M2, I1) and the control group was treated only with 0.03% isolates were characterized in a series of in vitro experiments Tween 80, using the following method. To each individual insect determining the effect of temperature on biological attributes such within a group, 0.4 lL of conidial suspension was topically applied as vegetative growth, sporulation and germination and a subset of to the abdominal region. Each group of treated insects were then isolates with the greatest promise as biological control agents were placed on to a M. paniculata leaf (three leaflets) in a 50 mm diam- selected (Orduño-Cruz et al., paper submitted). eter Petri dish. The Petri dish had a 30 mm diameter orifice in the Here we present the results from in vivo laboratory experiments lid covered with a mesh for ventilation and a damp filter paper disk on four of the selected isolates against adults of D. citri. Firstly we in the base. The filter paper was changed only once during the assessed the susceptibility of adult D. citri to infection by conidia of incubation period but 1 mL of distilled water was added every the selected isolates. Secondly, we tested the susceptibility of 3 days to maintain humidity. The petiole of the M. paniculata leaf D. citri to blastospores of two H. citriformis isolates. We then deter- was immersed in a 1.5 mL Eppendorf tube containing 1.5% mined whether the carriers used as standards to suspend conidia water-agar to maintain turgor pressure. All Petri dishes containing (distilled water or Tween 80) had an effect on germination of treated insects were incubated at 25 °C, 60% RH in a 16:8 light H. citriformis conidia compared with Metarhizium anisopliae regime. Mortality was recorded every 48 h for 16 days. The exper- (Metch.) Sorokin (Hypocreales: Clavicipitaceae) conidia. Finally, iment was carried out using a completely randomized design with we determined whether dry conidia of H. citriformis and five replicates, and the complete experiment was repeated on three M. anisopliae could achieve higher mortality rates compared to different occasions. those achieved using conidia or blastospores in suspension. 2.4. Statistical analysis
2. Material and methods Logistic regression, assuming a binomial distribution and allow- ing for over-dispersion where necessary, was used to assess 2.1. Production of adult D. citri for experiments
Adults of D. citri were obtained from a colony maintained on Table 1 List of isolates used in this study. Murraya paniculata (L.) Jack (Sapindales: Rutaceae) plants in a greenhouse at the Colegio de Postgraduados, Mexico. Original Species Isolate Host Geographical origin insects were collected in the locality of Cazones, Veracruz, B. bassiana B1 Diaphorina citri Veracruz, Mexico México in 2009, and have been maintained continuously since I. fumosorosea I1 Bemisia tabaci Yucatán, Mexico then. Eight-month-old M. paniculata plants were produced and M. anisopliae M2 Aeneolamia albofasciata Veracruz, Mexico H. citriformis H1 Unidentified parasitoid Yucatán, Mexico provided, on a monthly basis, by a private company (Ornaplant H. citriformis H2 Cicadellidae Yucatán, Mexico S.A. de C.V.) located in the Mexican state of Morelos. N. Orduño-Cruz et al. / Biological Control 90 (2015) 1–5 3 whether proportional mortality differed firstly between occasions 2.8. Infection of adults of D. citri by dry conidia of H. citriformis and M. on which the experiment was done, and secondly amongst fungal anisopliae isolates. No mortality was recorded in the control treatment, which was, therefore, excluded from the analysis. All statistical analysis We compared the pathogenicity of dry conidia of H. citriformis was performed using GenStat v. 8.0 (Payne et al., 2005). (H2) with dry conidia of M. anisopliae (M2) against adult D. citri using an autoinoculation approach. Adults of D. citri were exposed 2.5. Pathogenicity of H. citriformis blastospores to adult D. citri to yellow cardboard surfaces contaminated with either dry conidia of M. anisopliae or dry conidia of H. citriformis. Each yellow We compared the mortality caused by blastospores of two card (5 cm diameter) was covered in a thin layer of molten SDBY H. citriformis isolates (H1 and H2) against adult D. citri. and inoculated with conidia of either M. anisopliae (0.01 g) or Blastospores were produced by adding four 5 mm diameter plugs H. citriformis (0.10 g); conidia were evenly distributed over the sur- cut from the growing edge of 40 days old cultures to 50 mL of face using a sterile fine brush. The yellow cards were then placed in Sabouraud Dextrose Broth supplemented with 3% yeast extract a 5 cm diameter Petri dish with a M. paniculata leaf as described (SDBY) in a 250 mL Erlenmeyer flask. Flasks were incubated on a previously. Then, 22 adult D. citri were introduced into each dish rotary shaker at 250 rpm and 25 °C for 8 days. The concentration by placing them on the M. paniculata leaf. Dishes were incubated of blastospores in the medium was estimated as described previ- at 25 °C, 60% RH in a 16:8 light regime for 6 days, and mortality ously, and then adjusted to achieve 1  107 blastospores mLÀ1 by was recorded every 24 h. After 24 h of incubation, 10 adults from the addition of 0.03% Tween 80. For each repetition in time, one each treatment were removed and placed into 1.5 mL Eppendorf flask per isolate was used. Replicate groups of 20 adults were inoc- tubes containing 100 lL of 0.03% Tween 80, vortexed for 2 min to ulated with 1  107 blastospores mLÀ1 suspensions of each fungal remove conidia from the insects’ bodies, and the number of conidia isolate and the control group was treated only with 0.03% Tween estimated using a haemocytometer. Mortality, therefore, was 80. The inoculation method, incubation conditions, mortality recorded based on 12 insects per treatment. The experiment was recording, statistical design, replication and analysis were as done using a completely randomized design, with four replicates described previously for the experiment with conidial suspensions. per treatment  isolate combination, and the complete experiment was repeated on three separate occasions.
2.6. Effect of carrier solution on the germination of H. citriformis and 2.9. Statistical analysis M. anisopliae conidia
Mortality data were not analyzed as 100% infection was Conidia of H. citriformis (H2) and M. anisopliae (M2) were either achieved in all treatments and for both isolates. Only the number suspended in 0.03% Tween 80 or distilled water, or were left as dry of conidia of each isolate that were attached to the insect’s body conidia (not suspended in any aqueous solution) using the follow- were compared, and these data did not require transformation ing procedures. One 8 mm diameter plug was cut from the growing prior to analysis. Analysis of variance (ANOVA), with blocking edge of a 15 or 40-day-old culture of M. anisopliae and H. citriformis structure for occasion was used to assess whether there was an respectively. Each plug was placed into 5 mL of either distilled effect of isolate on the number of conidia attached to the insect´s water or 0.03% Tween 80 in 50 mL centrifuge tubes and vortexed body. All statistical analysis was performed using GenStat v. 8.0 for 5 min. Ten microliter aliquots of 1 Â 105 conidia mLÀ1 suspen- (Payne et al., 2005). sions were placed at the center of SDA plates and germination assessed after 24, 36 and 48 h. For the dry conidia treatment, a sterile fine brush was used to remove an unquantified number of 3. Results conidia from 15 and 40 days old colonies of M. anisopliae and H. citriformis respectively, and deposit them on to SDA plates; the 3.1. Susceptibility of adults of D. citri to infection by four selected conidia were brushed over the complete surface of the medium. isolates For all isolates, a different plate was prepared for each assessment time (24, 36 and 48 h). All SDA plates were incubated at 25 °C, 60% No differences were found amongst the occasions that the RH and a 16:8 light regime. After the appropriate incubation time, experiment was repeated (F2,42 = 1.60, P = 0.215); therefore, data conidia were fixed with 10% cotton blue in lactophenol prior to from all repetitions were combined for further analysis. determining the proportion germinated. At each assessment time, Significant differences were found amongst isolates (F3,42 = 100 conidia were randomly selected in each plate and the propor- 103.40, P = 0.001), where the isolate causing the greatest mortality tion germinated recorded; conidia were considered as germinated when the length of the germ tube was at least the same as the width of the conidium. The experiment was done using a com- pletely randomized design, where each combination of incubation time (3) Â isolate (2) Â type of suspension (3) was replicated four times, and the complete experiment was repeated on three sepa- rate occasions.
2.7. Statistical analysis
Data (number of conidia germinated out of 100 expressed as a proportion) for the two suspensions and dry conidia (treatments) were analyzed using linear mixed models with treatments, isolate and incubation times as effects (and interactions) and variance components estimated using the restricted maximum likelihood Fig. 1. Proportion of Diaphorina citri adults infected by isolates of B. bassiana (B1), method (REML). All statistical analysis was performed using M. anisopliae (M2), I. fumosorosea (I1) and H. citriformis (H2). Error bars represent GenStat v. 8.0 (Payne et al., 2005). 95% confidence intervals back-transformed from the logistic scale. 4 N. Orduño-Cruz et al. / Biological Control 90 (2015) 1–5 was M2 (M. anisopliae), followed by I1 (I. fumosorosea), which both caused mortalities above 95%. These were followed by isolate B1 (B. bassiana) and finally isolate H2 (H. citriformis) where mortality was below 40% (Fig. 1).
3.2. Pathogenicity of H. citriformis blastospores against adult D. citri
No differences were found amongst the occasions that the experiment was repeated (F2,20 = 0.12, P = 0.888); therefore, data from all repetitions were combined. No differences were found between the two H. citriformis isolates evaluated (F1,20 = 0.27, P = 0.606). The mortality caused by isolates H1 and H2 was 61.6 and 65% respectively.
3.3. Effect of the carrier solution on germination of H. citriformis and Fig. 3. Mean number of conidia from the isolates M2 and H2 that were attached to M. anisopliae conidia adult D. citri. Error bars represent ±1 Â SEM.
Analysis of germination data showed a significant effect of all are known to reduce the feeding activity of infected hosts (Avery treatments, isolate, incubation times and all interactions amongst et al., 2009), and as a consequence, transmission of HLB might be them (P > 0.001). Overall, isolate M2 achieved 100% germination reduced, but this needs further experimental confirmation. The in all treatments and all germination times, except after 24 h of I. fumosorosea isolate (I1) we evaluated caused similar levels of incubation of dry conidia, where germination was 95% (Fig. 2). mortality in D. citri adults as the isolates of B. bassiana (B1) and Conidia of isolate H2 achieved a maximum of 15% germination in M. anisopliae (M2), suggesting that other attributes should also Tween and distilled water treatments at all incubation times; how- be considered in the selection process. Isolates of B. bassiana and ever, dry conidia achieved more than 50% after 24 h of incubation M. anisopliae have been evaluated against D. citri previously, with reaching 95% germination after 48 h of incubation (Fig. 2). variable results of 75% and 30% mortality respectively (Padulla and Alves, 2009) and 50% and 60% mortality respectively 3.4. Infection of adults of D. citri by dry conidia of H. citriformis and M. (Lezama-Gutiérrez et al., 2012). It is difficult to explain the differ- anisopliae ences between our results and those reported previously, but it strongly suggests that local isolates should be considered when All adults of D. citri exposed to dry conidia of either M2 or H2 selecting a biological control agent. resulted in 100% infection. The number of conidia attached to the It was surprising that H. citriformis isolate H2, which came from insect´s body was different between isolates (F1,56 = 49.34, an infected D. citri adult, did not cause greater than 50% mortality 5 P = 0.001). A mean of 3.8 Â 10 conidia per insect was recorded when inoculated as conidia in suspension, unlike the other three 5 for isolate H2, and 7.5 x 10 conidia per insect for M2 (Fig. 3). isolates (Fig. 1); this was particularly interesting as H. citriformis is the most common species infecting D. citri adults in the field 4. Discussion (NOC, personal observation). However, when D. citri adults were exposed to dry conidia, 100% mortality was achieved within 6 d., Our data provides evidence that three of the four isolates which was similar to previous reports where inoculations were assessed (B1, M2 and I1) had great potential to be used for biolog- made using dry conidia (Meyer et al., 2007; Casique-Valdes et al., ical control of adult D. citri in an inundation approach where coni- 2011). It is possible that the greater number of dry conidia 5 dia are applied in suspension. Our results with I. fumosorosea were attached to the insects (4 Â 10 conidia per insect, Fig. 3), com- 4 in line with the studies of other researchers who also reported high pared to the conidial suspension inoculated (4 Â 10 conidia per mortality (100%) (Hoy et al., 2010; Avery et al., 2011). Although the insect), may have contributed to this greater mortality; however, speed of kill caused by an entomopathogenic fungus is not compa- we believe that the drastic reduction in germination caused by rable with that of a chemical insecticide, entomopathogenic fungi the carriers used to maintain conidia in suspension had the most serious negative effect (Fig. 2). Although we cannot provide exper- imental evidence for the mechanism, we believe a number of dif- ferent factors could be contributing to the failure of H. citriformis conidia to germinate when in suspension. It is likely that the mucilaginous outer layer, mainly protein (40%) and sugar (10%), of H. citriformis conidia (Rosas-Acevedo et al., 2003) could be dis- solved, and with this, the conidia would be exposed to desiccation and to the toxic polyphenols that are likely to be present in the insect´s cuticle (Boucias et al., 1998). It is clear from our results that using a dry powder of H. citriformis, has greater potential than using conidia in suspension. With our culture conditions on SDA, H. citriformis took 40 days of incubation before conidia were produced. Blastospores can be produced much faster than this (8 days of incubation), and, in the case of I. fumosorosea blastospores, have proved promising for control of the whitefly Trialeurodes vaporariorum Westwood in the field (Kim et al., 2013). For these reasons we evaluated Fig. 2. Proportion of conidia germinated for the isolates M2 and H2 when suspended in 0.03% Tween 80, distilled water or as dry conidia (not suspended). H. citriformis blastospores against D. citri and achieved mortality Error bars represent ±1 Â SEM. of 61.6% and 65% for the two isolates evaluated respectively, which N. Orduño-Cruz et al. / Biological Control 90 (2015) 1–5 5 was greater in comparison with the previous experiment where entomopathogenic fungus Isaria fumosorosea (Hypocreales: Cordycipitaceae) only 40% mortality was achieved with conidial suspensions of iso- under laboratory conditions. Florida Entomol. 92, 608–618. Avery, P.B., Wekesa, V.W., Hunter, W.B., Hall, D.G., McKenzie, C.L., Osborne, L.S., late H1 (Fig. 1). We believe that this may be because blastospores Powell, C.A., Rogers, M.E., 2011. Effects of the fungus Isaria fumosorosea germinate faster than conidia, as demonstrated previously for (Hypocreales: Cordycipitaceae) on reduced feeding and mortality of the Asian I. fumosorosea (Vega et al., 1999). citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae). Biocontrol Sci. Tech. 21, 1065–1078. In our autoinoculating device we used yellow cardboard in Boucias, D.G., Farmerie, W.G., Pendland, J.C., 1998. Cloning and sequencing of cDNA order to attract the insects to the conidia; this was a similar of the insecticidal toxin Hirsutellin A. J. Invertebr. Pathol. 72, 258–261. method to that used by Avery et al. (2009) who used blastospores Bové, J.M., 2006. Huanglongbing: a destructive, newly-emerging, century-old disease of citrus. J. Plant Pathol. 88, 7–37. of I. fumosorosea on yellow card and achieved almost 100% mortal- Casique-Valdes, R., Reyes-Martinez, A.Y., Sanchez-Pena, S.R., Bidochka, M.J., Lopez- ity in D. citri within 8 days. Using a similar approach, Moran et al. Arroyo, J.I., 2011. Pathogenicity of Hirsutella citriformis (Ascomycota: (2011) achieved 70% and 40% mortality in D. citri in laboratory and Cordycipitaceae) to Diaphorina citri (Hemiptera: Psyllidae) and Bactericera cockerelli (Hemiptera: Triozidae). Florida Entomol. 94, 703–705. greenhouse experiments respectively. However, these authors Doutt, R.L., Smith, R.F., 1971. 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