The citrus rust mite, Phyllocoptruta oleivora Ashmead (Acari: Eriophyidae), is a major pest of citrus, affecting both quality and yield. Although entomopathogenic fungi such as Beauveria bassiana (Bals.-Criv.) Vuill. (Cordycipitaceae), Metarhizium anisopliae (Metschn.) Sorokīn (Clavicipitaceae), and Isaria fumosorosea Wize (Cordycipitaceae) have been used for insect and mite control, the last 2 have never been applied to control citrus rust mite. The aim of this study was to evaluate the effect of 3 concentrations of B. bassiana, M. anisopliae, and I. fumosorosea on mortality and growth rate (r) of P. oleivora populations under greenhouse conditions. The fungi were isolated from agricultural soil samples. Mobile stages of P. oleivora were collected from leaves of commercial Valencia orange groves. At a concentration of 1 × 108 conidia mL-1, B. bassiana and M. anisopliae caused > 60% mortality in P. oleivora populations, whereas mortality due to I. fumosorosea never exceeded 50%. There was a significant inverse relationship between the percent mortality and the LT50. Application of B. bassiana resulted in the slowest growth rate of P. oleivora populations, which was 10.5 × slower than the control population receiving no fungus application. Our results demonstrate that B. bassiana and M. anisopliae are efficient in control of P. oleivora populations under greenhouse conditions, whereas I. fumosorosea produces moderate control.
The citrus rust mite, Phyllocoptruta oleivora (Ashmead) (Acari: Eriophyidae), is a pest of citrus in tropical regions, where environmental conditions are optimal for population growth (Childers et al. 1996; Aghajanzadeh & Mallik 2007; Rogers & Stansly 2016). Together with the Mexican fruit fly, Anastrepha ludens Loew (Diptera: Tephritidae), P. oleivora contributes to severe phytosanitary problems for citrus production in the state of Tamaulipas in northeastern Mexico (Varela-Fuentes et al. 2005; Varela-Fuentes et al. 2013; Vanoye-Eligio et al. 2017). Phyllocoptruta oleivora can infest leaves and green branches of citrus.
Damage occurs when P. oleivora feed on the developing fruit; penetration of epidermal cells by the mite's stylet causes dark brown areas to develop on the fruit surface (McCoy & Albrigo 1975). Citrus rust mites are present throughout the year, selecting fruits that are exposed to sunlight, but they avoid the heat in the outer portions of the fruit that get very hot from exposure to sunlight (Varela et al. 2005; Varela-Fuentes et al. 2013). Heavy infestation with P. oleivora reduces the size and weight of fruit; this reduces juice volume, increases acidity and soluble solids, and reduces the aesthetic appearance of the fruit, limiting their marketability (Varela et al. 2005; Imbachi et al. 2012; Mesa & Rodríguez 2012; Varela-Fuentes et al. 2013).
Throughout Mexico, including northeastern Mexico, the principal control method for P. oleivora is the use of broad spectrum insecticides (Alves et al. 2005). However, this practice increases the risk of insecticide resistance and environmental degradation of agroecosystems (Varela-Fuentes et al. 2013). The use of entomopathogenic fungi for pest control is an alternative strategy, particularly for the problems of resistance (Omoto et al. 1994) and environmental pollution, thus improving the economic and biological sustainability of agroecosystems (Alves et al. 2004).
The majority of fungi that contribute to natural regulation of insect and mite populations are from the orders Hypocreales and Entomophthorales (Dolinski & Lacey 2007). The latter are difficult or impossible to mass-produce, and therefore have not been produced commercially or applied on a large scale. In contrast, several species of Hypocreales are commercially available for control of insect and mite pests; some of these belong to the genera Beauveria, Metarhizium, Isaria, Aschersonia, Hirsutella, and Lecanicillium (Alves 1998; Inglis et al. 2001; Goettel et al. 2005).
The fungi Beauveria bassiana (Bals.), Metarhizium anisopliae (Metschinkof), and Isaria fumosorosea (Wize) have been studied widely as biological control agents, including as mycoacaricides (Tamai et al. 1999, 2002; Amjad et al. 2012; El-Sharabasy 2015). There have been a few studies on the efficacy of B. bassiana to control P. oleivora under laboratory conditions (Alves et al. 2005); despite this, there have been no studies under greenhouse and field conditions. Similarly, no studies have been conducted on M. anisopliae to control P. oleivora. Paecilomyces fumosoroseus (= Isaria fomosorosea) (Wize) A. H. S. Br. & G. Sm. (Cordycipitaceae) has been evaluated to control 2 other mites, the citrus brown mite, Eutetranychus orientalis (Klein) (Prostigmata: Tetranychidae), and the 2-spotted spider mite, Tetranychus urticae Koch (Prostigmata: Tetranychidae) under laboratory and greenhouse conditions, respectively (Luangsa-Ard et al. 2005; Kim et al. 2008; El-Sharabasy 2015).
Alternative control strategies for P. oleivora that contribute to economic gain through effective pest control and reducing resistence, and are environmentally friendly, are urgently required. In the present study we evaluated mortality and population growth rate of mobile stages of P. oleivora following application of different concentrations of 3 entomopathogenic fungi: B. bassiana, M. anisopliae, and I. fumosorosea. We also determined lethal times for these entomopathogens to kill 50% of citrus rust mite.
Materials and Methods
greenhouse study was done at the Institute of Applied Ecology, Autonomous University of Tamaulipas, Victoria, Tamaulipas, Mexico. Eighty Citrus sinensis (L.) Osbeck (Rutaceae) variety ‘Valencia’ plants that were certified free of pests and diseases were obtained from the Francisco Villa Experimental Facility (23.897222°N, 99.131944°W), Güemez, Tamaulipas, Mexico. Plants were 65 ± 5 (average ± SD) cm in height and were placed in the greenhouse in pots at a distance of 30 cm apart. Environmental conditions in the greenhouse during the experiment were 35 ± 5 °C, 80 ± 10% RH, and a 11:13 (L:D) photoperiod. Plants were irrigated every 2 d with 200 mL of water, and fertilized once using HUMIMAX (Humic substances derived from leonardite 12%, soluble potassium 2%, amino acids 0.50%, manganese 3%, iron 3%, zinc 1%, boron 0.50%, moisturizers, dispersing and penetrating 78%) at a dose of 2.5 mL per L of water. Fertilization was performed 20 d before the infestation to avoid any effect on the mites.
COLLECTION OF MOBILE STAGES OF PHYLLOCOPTRUTA OLEIVORA AND ESTABLISHMENT ON EXPERIMENTAL TREES IN THE GREENHOUSE
Mobile mite stages were collected from leaves of commercial Valencia orange groves from 3 localities: Mártires Chinameca (24.096111°N, 99.140555°W), Úrsulo Galván (24.093611°N, 99.136666°W), and La Misión Victoria (23.812500°N, 99.159444°W) in Tamaulipas State, Mexico. Collections were made in Mar and Apr 2017 when we predicted that the mite populations were likely to be high (Landeros et al. 2003). We selected mature and immature leaves and fruits that were showing symptoms of damage by P. oleivora. Samples were collected into brown paper bags, placed inside iceboxes at 5 ± 1 °C, and transported to the laboratory at Institute of Applied Ecology, Autonomous University of Tamaulipas. The presence of mites on samples was confirmed using a stereoscopic microscope (UNICO Stereo & Zoom Microscopes ZM180, Dayton, New Jersey, USA).
Colonies of mites were established on all trees in the greenhouse over a period of 18 d; every 2 d a leaf disc containing 10 to 12 mites was placed at the top of each tree (9 infested leaf discs added in total to every tree). Branches with infested leaves were labelled for subsequent monitoring. Two d before the fungal applications, 1 leaf was taken from each tree and from trees being allocated to different treatments, and the numbers counted under a microscope to confirm that mite colonization had been achieved, and the count was taken to be the initial population of mites (X0); after observing the infested leaves, they were returned to the trees from which they had been sampled.
SOURCE AND PRODUCTION OF ENTOMOPATHOGENIC FUNGI
Beauveria bassiana, M. anisopliae, and I. fumosorosea were isolated from agricultural soil samples according to Sánchez-Peña et al. (2011) and grown on Potato Dextrose Agar (PDA, DIBICO, Mexico City, México) in 90 × 15 mm diam Petri dishes for 21 d at 25 ± 2 °C, 70% RH, 12:12 h (L:D) photoperiod. Isolates were identified using a light microscope (Standard 25 ICS, Carl Zeiss AG, Oberkochen, Germany) (Humber 1997). Under sterile conditions, conidia from each isolate were scraped into sterile tubes of 50 mL 0.02% Tween® 20 aqueous solution to produce a suspension; the conidial concentration was estimated using a Neubauer hemocytometer (Fisher Scientific, Pittsburgh, Pennsylvania, USA), adjusted to 1 × 108 conidia mL-1, and used to inoculate 1 of the 250 mL flasks (Pyrex, New York, USA), each containing 100 mL of Sabouraud Dextrose Broth (Bioxon, New Jersey, USA); 10 mL of suspension was added to each flask. The flask cultures were incubated at 28 °C and shaken manually every third d. After 5 d, 30 mL from the flask cultures was inoculated onto sterile rice (Oryza sativa L.) (Poaceae) in polyethylene bags: 200 g of rice sterilized in 100 mL water per bag (capacity = 1 kg) inoculated with 30 mL aliquots of suspension. The bags of rice were incubated for 14 d under ambient conditions (25–28 °C, 12:12 h [L:D] photoperiod). After 15 d of incubation, the conidia were harvested by adding 100 mL of 0.02% Tween® into each bag of rice, which was then shaken. The solution was filtered through a sieve (500 μm, Gilson®) to recover the liquid. Conidial concentration was estimated using a Neubauer hemocytometer and adjusted to provide 1 × 108, 5 × 107, and 1 × 107 conidia per mL-1 suspensions of each isolate for the greenhouse experiment.
The experiment was carried out in the following manner: 10 to 12 mites were released 2 days before the application of the fungus. Three leaves of 6 randomly sampled trees were taken for treatment, and the number of mites counted from those leaves were called the initial population (X0). Further, initial mite count determined the homogeneous distribution and establishment of the P. oleivora population within the greenhouse.
After the application of fungus, the study was carried out using 1-way designs per counting date. The structure of this design is a set of t treatments, also called a unifactorial treatment structure. Each way was designed to analyze the effects of concentrations of each fungus per count. Three replicates (leaves) were counted for each treatment, analyzing 36 replicates per count date. The sample unit was the number of mites on the leaves. For each concentration, 6 trees were randomly used. Each treatment was separated by 2 m to avoid cross contamination. Each treatment tree was sprayed with 0.5 L of the relevant fungal species and concentration using a Truper® manual sprayer (Model 14687; Truper®, Victoria City, Tamaulipas, Mexico). The control treatment (n = 18 trees total) were sprayed with only the carrier Tween® without conidia. The counts were made at 3, 5, 7, and 10 d after the application of each treatment. The mortality criterion was determined by mites with evidence of mycelial growth, which was determined by observation under the stereoscope. Also, live mites were counted to obtain their growth rate. After each count, the leaves were destroyed (total of 36 leaves per treatment per concentration combination and control for each sampling occasion). Environmental conditions in the greenhouse during the experiment were 35 ± 5 °C, 80 ± 10% RH, and 11:13 h (L:D) photoperiod.
The Henderson and Tilton (1955) formula was used to correct the data for control population size, and to establish the percentage of mortality for each treatment compared with the control. This was necessary because in the absence of treatment (the control) mite populations may still fluctuate (increase or decrease) due to the biology of the mite or the quality of food.
Arccosine transformation was used to stabilize variance in population counts. Data on mortality counts were then analyzed by ANOVA and the mean comparisons using Tukey's hsd test (P < 0.05). The first count of live individuals was analyzed by 1-way ANOVA, and thereafter counts were analyzed by 2-way ANOVA. The mean lethal time values (LT50) were calculated using Probit analysis (Finney 1971). SAS software was used for all analyses (SAS 2002).
Virulence and lethal mean time (LT50) of entomopathogenic fungi to control mobile stages of Phyllocoptruta oleivora.
Population growth rates in different treatments were estimated using the equation proposed by Odum (1971), r = (1/t)(X t /X 0), and used by Soler-Salcedo et al. (2006) and Chacón-Hernández et al. (2017), where t is the time in d, X 0 and X t the number of mites at time 0 (or initial number of individuals), and the number of mites at time t at the end of the next sampling period, respectively. The units of this parameter represent the number of offspring that each individual produced per d throughout the study period and is expressed as d-1 (Soler-Salcedo et al. 2006).
We evaluated the effect of applications of B. bassiana, M. anisopliae, and I. fumosorosea at 3 concentrations on populations of the mobile stages of P. oleivora in the greenhouse (Table 1). On d 3 after application there was a significant effect of concentration on the number of mobile stages of P. oleivora (F 9, 20 = 27.73; P < 0.0001). At this time, mortality ranged between 21.01% (I. fumosorosea 5 × 107) and 27.71% (B. bassiana 1 × 108). On d 5, significantly higher mortality was achieved in the B. bassiana 1 ×108 conidia mL-1 treatment compared with all the other treatments (F 9, 20 = 180.08; P < 0.0001). By d 7, the greatest mortalities were seen in the B. bassiana 1 × 108 conidia mL-1 and M. anisopliae 1 × 108 conidia mL-1 treatments (F 9, 20 = 9.54; P < 0.0001). By d 10, the greatest mortalities also were found in the B. bassiana 1 × 108 conidia mL-1 and M. anisopliae 1 × 108 conidia mL-1 treatments (F 9, 20 = 125.87; P < 0.0001). All concentrations of I. fumosorosea caused moderate mortality in P. oleivora (Table 1).
The time for 50% mortality (LT50) values ranged from 5 to 23 d; B. bassiana had shorter LT50s than M. anisopliae and I. fumosorosea, but asymptotically, concentrations were significantly different according to 95% CI. The trend for LT50 values indicate a significant negative relationship with concentration, i.e., the higher the concentration of conidia mL-1, the shorter the mean lethal time (Table 1).
The initial densities of P. oleivora before application of entomopathogenic fungi were not significantly different among treatments (F 3, 8 = 0.39; P > 0.05) (Fig. 1), confirming that populations had established homogenously inside the greenhouse. After application of entomopathogenic fungi, there were significant differences in population sizes of live mites among treatments (F 9, 80 = 31.75; P < 0.0001), on fluctuating population of P. oleivora (F 3, 80 = 99.68; P < 0.0001), and there was a significant interaction (F 27, 80 = 6.69; P < 0.0001). Thus, populations of live mobile stages of P. oleivora in the control increased in number more than in all the fungal treatments. Populations rapidly increased on control trees in the first 5 d and remained steady thereafter. Individuals in control populations had a mean offspring production rate of 0.42 d-1; in the B. bassiana 1 × 108, 5 × 107, and 1 × 107 conidia mL-1 treatments, it was 0.04, 0.04, and 0.06 d-1, respectively; in the M. anisopliae 1 ×108, 5 × 107 and 1 × 107 conidia mL-1 treatments, it was 0.07, 0.08, and 0.08 d-1, respectively; in the I. fumosorosea 1 × 108, 5 × 107, and 1 × 107 conidia mL-1 treatments, it was 0.09, 0.10, and 0.17 d-1, respectively (Fig. 1). The population fluctuations observed over the first 3 d suggest that mortality was occurring most rapidly in the B. bassiana treatments, presumably due to infection.
The overall population growth rate was lowest in the 3 B. bassiana treatments (all 3 concentrations) due to higher fungus-related mortality. The opposite was apparent in the I. fumosorosea treatments, presumably because it was less pathogenic to P. oleivora. Metarhizium anisopliae was intermediate in its effect on population growth (Fig. 1, Table 1). There was a dose-related effect of different concentrations of all 3 fungi on P. oleivor population growth rates; at 1 × 108, 5 × 107, 1 × 107 conidia mL-1 of B. bassiana, growth rates were 10.5, 10.5, and 7.0, respectively, slower than the control; for M. anisopliae they were 6.4, 5.6, and 5.0 slower, respectively; and for I. fumosorosea they were 4.6, 4.2, and 2.5, respectively, slower. This indicates that B. bassiana is more effective in controlling populations of citrus rust mite under greenhouse conditions than the other 2 fungal species.
Mortality in the mobile stages of the citrus rust mite caused by entomopathogenic fungi increased with time after application. Beauveria bassiana caused the greatest mortality in populations of P. oleivora between d 3 and the end of the experiment. This fits with the fact that B. bassiana requires only 24 h to colonize its host (Alves et al. 2005). Lower concentrations of each fungus would reduce the numbers of hosts killed, and accounts for the dose response we observed.
The mortalities observed were less than in studies of P. oleivora by Alves et al. (2005), who evaluated B. bassiana on citrus rust mite, whereas Barreto et al. (2004) and Bugeme et al. (2008) evaluated B. bassiana and M. anisopliae on Mononychellus tanajoa Bondar and Tetranychus evansi Baker and Pritchard (both Prostigmata: Tetranychidae), respectively, and Zemek et al. (2016) evaluated I. fumosorosea to control Tetranychus urticae Koch (Acari: Tetranychidae), where they found mortality above 90%, at 5, 8, 10, and 15 d after application. This may be due to differences in environmental conditions in the studies. In our study, the temperature was 35 ± 5 °C and RH was 80 ± 10%, whereas the other studies were done under laboratory conditions of 25 ± 2 °C, and 70 to 98% RH. Bugeme et al. (2008) state that mortality varies with species of entomopathogenic fungus, isolates, and temperature. The same authors found that B. bassiana and M. anisopliae also provided good control of the red spider mite, T. evansi, at temperatures of 30 ± 5 °C. Furthermore, it has been reported that several isolates of fungi are pathogenic to arthropod pests at temperatures ranging from 25 to 35 °C (Hsiao et al. 1992; Fargues & Remaudiere 1977; Thomas & Jenkins 1997; Ekesi et al. 1999; Milner et al. 2003; Dimbi et al. 2004; Cuthbertson et al. 2005; Bugeme et al. 2008; Mishra et al. 2015).
In our study, all concentrations of I. fumosorosea achieved mortalities of less than 50% in 10 d, which was lower than figures reported by Zemek et al. (2016), who found that at a concentration of 4 × 107 conidia mL-1, I. fumosorosea caused 92.1% of mortality of T. urticae females in laboratory experiments, whereas Chandler et al. (2005) reported mortalities of 21 to 22% and 97% (concentrations 1 × 107 and 1 × 108 conidia mL-1, respectively) of mobile stages of T. urticae on tomato Lycopersicon esculentum Miller (Solanaceae) under laboratory and greenhouse conditions, respectively.
Environmental factors, such as temperature and humidity, affect not only the survival and virulence of entomopathogenic fungi, but also influence host-pathogen interactions (Kiewnick 2006). Optimal temperature and humidity requirements for survival and virulence vary among different fungal isolates (Fargues & Remaudiere 1977). Beauveria bassiana is effective over a wide temperature range (25–35 °C) under favorable humidity conditions (90% RH) (Bugeme et al. 2008; Mishra et al. 2015). The efficiency of entomopathogenic fungi is influenced by temperature and relative humidity (Mishra et al. 2015), and thus was reflected in the LT50, since these environmental factors in this study ranged between 35 to 40 °C, and 70 to 90% R.H.
In concentrations of 1 × 108, 5 × 107, 1 × 107 conidia mL-1 of B. bassiana, the LT50s achieved were 5.05, 6.47, and 6.04 d, respectively, and for M. anisopliae the values achieved were 5.64, 6.98, and 6.54 d, respectively. This is comparable with reports of Bugeme et al. (2008), who found that at a concentration of 1 × 107 conidia.mL-1 of B. bassiana isolates ICIPE278 and ICIPE279, LT50s of 1.9 and 2.0, respectively, were achieved at 35 °C to control adult female populations of T. evansi. In contrast with a concentration of 1 × 107 conidia per mL, isolates ICIPE55, ICIPE59, and CIPE78 of M. anisopliae achieved LT50s of 2.6, 3.4, and 2.7 d, respectively, to control T. evansi at 35 °C, which was faster. In our study, LT50 can be correlated with mortality rate (r = −0.8976; P < 0.001), which implies that high mortality and short LT50s are linked, and that mortality is related to concentration, i.e., the infection rates are dependent on conidial concentrations. The LT50s obtained showed that with increasing concentrations (1 × 107, 5 × 107, and 1 × 108), the decrease in population size of citrus rust mite was more evident; however, the population growth rate (r) is reduced, and the reproductive potential of the females is possibly reduced, decreasing the fertile period and the oviposition time.
The present study revealed that B. bassiana and M. anisopliae are efficient in controlling P. oleivora populations under greenhouse conditions, whereas I. fumosorosea produces moderate control. The mortality of the population of mobile stages of P. oleivora depends on the isolate concentration and fungus. Also, the higher the concentration, the higher the mortality in the citrus rust mite population. Our results demonstrate that entomopathogenic fungi can be used as a potential microbial control on P. oleivora in a program of integrated pest management (IPM) to control citrus rust mite in the orange crop under greenhouse conditions.
We thank ‘Programa para el Desarrollo Profesional Docente para el Tipo Superior’ for financial support of project DSA/103.5/16/10526.