In vitro pathogenicity of northern Peru native bacteria on Phyllocnistis citrella Stainton ( Gracillariidae : Phyllocnistinae ) , on predator insects ( Hippodamia convergens and Chrisoperna externa ) , on Citrus aurantifolia Swingle and white rats

In Peru, the leaf miner Phyllocnistis citrella attacks citrus crops, including the economically important species Citrus aurantifolia, adversely affecting production. The objective of this work was to determine the in vitro pathogenic ability of enterobacteria isolated from within P. citrella. In addition, the pathogenic effects of these enterobacterias were tested on the predator insects Hippodamia convergens and Chrisoperna externa, on the host plant C. aurantifolia and on rats. The insects were captured in plantations of C. aurantifolia in the Piura Region. Phyllocnistys citrella was the most frequently occurring pest (98%), among other identified pests. From diseased larvae of P. citrella, the bacteria Serratia sp., Pseudomonas sp., and Enterobacter aerogenes were isolated. The three bacterial species had a similar pathogenic effect on P. citrella after 48 h (74.1% average mortality). Serratia sp. caused the highest mortality after 24 h in H. convergens (40%) and C. externa (30%), whereas the Lowest mortality rates were induced at 72 h by E. aerogenes on C. externa (3%) and by Pseudomonas sp. on H. convergens (10%). The bacteria did not affect neither C. aurantifolia or the rats, which gained the same weight as control animals. Additional key words: Enterobacter, entomopathogenic bacteria, Piura, plant health and crop protection, Pseudomonas, Serratia.


Introduction
Key lime, Citrus aurantifolia (Rutaceae), is an important agricultural crop in Peru, both for export and domestic consumption.Citrus aurantifolia plantations cover 10,528 ha or 38.6% of the area dedicated to orchards in the Piura Region.Citrus leaf miners (Phyllocnistis citrella), can limit or reduce citrus fruit production (Ginocchio, 1993).This pest attacks citrus trees by infesting the buds and decreasing the leaf area.They reduce the leaf photosynthetic rate and cause them to roll up and drop off (Schaffer et al., 1997).Larvae of P. citrella can eat 1 to 7 cm 2 of leaf per day.
The pathogenic bacteria, Pseudomonas sp., isolated from diseased larva of P. citrella induced 80% mortality on this pest in micro-plot field bioassays (Sepúlveda et al., 2001).Pseudomonas aeruginosa was pathogenic against the orthopterans Melanoplus bivittatus and Cammula pellucida; this bacterium does not normally multiply in the digestive tract of insects, but it can in the haemocoel (Angus, 1965).In a similar study, (Goptal and Gupta, 2002), detected high concentrations (10 9 -10 10 cells mL -1 ), of Pseudomonas alcaligenes in the haemolymph of dead Oryctes rhinoceros grubs, a tropical coconut pest.Approximately 52% of the grubs succumbed to septicaemia.Although P. alcaligenes is a normal bacterial component in the gut of healthy grubs, under other conditions, it can be an opportunistic pathogen.The main problem with this pathogen has generally been its adaptation and survival when used in new environments (Ohba and Aizawa, 1986).Therefore, the origin of a control agent is important.
The bacterial genus Serratia consists of ten recognized species; one group is an important nosocomial pathogen and the other species cause less frequent infections (Carrero et al., 1995).However, S. marcescens has been isolated from the haemolymph of boll weevils (Anthonomus grandis) (Schmitz and Braun, 1985) and strains of Serratia have been isolated from different soils and the gut of invertebrates (Ashelford et al., 2002).S. marcescens isolated from boll weevils can cause disease in guinea pigs (Cavia sp), mice (Mus musculus) (Lyerly and Kreger, 1983), and insects by causing evolution of exoproteases during pathogenesis (Stock et al., 2003).Serratia proteamaculans, isolated from the spider Nephilia clavata, could have mutualistic or synergic relationships with exoprotease production by the spider in order to digest its victims.However, this does not exclude the possibility that the bacterium could also be pathogenic to the spider as well as the insects (Dece-due et al., 1979).Serratia entomophila and S. proteamaculans cause amber disease in grass grub Costelytra zealandica (Coleoptera); S. entomophila has been isolated from insects and the environment, but not from animals other than insects.A product based on S. entomophila has been successfully developed to control the pest C. zealandrica (O' Callaghan and Gerard, 2005).
The aim of this work was to determine the in vitro pathogenic ability of enterobacteria isolated from P. citrella on this pest; this was compared with the pathogenic effects of the bacteria on the predator insects, the convergent ladybird, Hippodamia convergens, and Chrisoperna externa, on the host plant Citrus aurantifolia, and with rats.

Location of the study
The Piura Region is located on the northwest coast of Peru between 3º and 7º S. It is divided into eight provinces, including Sullana (4°52´49"S, 80°41´07"W, elevation 70 m) and Chulucanas (05°05´57"S, 80°41´07"W, elevation 115 m).Ecologically, the Piura region is divided between tropical dry forest in the north and the Sechura desert in the south.The northern part of the Sechura desert has an annual temperature of 26ºC and an average annual rainfall of 50 mm concentrated in the months of January, February, and March.In the study area, winters are hot and humid with temperatures of 28ºC and the maximum average temperature exceeds 35ºC.The average temperature during the region's dry summers ranges between 24 o and 20ºC.

Estimation of the Phyllocnistis citrella population
The relative abundance of P. citrella larvae was determined over 9 months on a 1 ha plantation of C. aurantifolia, plantation at the Fundo Tungazuca farm in Cieneguillo Village, Sullana Province.Phyllocnitis citrella and the other insect pests of C. aurantifolia deposit their eggs on C. auranitfolia leaves or are found as pupa and larvae in leaf galleries.These leaves can be collected with the insects inside or on them.Special insect catching traps were not necessary.Other common insects such as bees (Apis sp.) and predators were easy to catch with nets.Insects from 10 branches from each cardinal point of different citrus trees were collected.
All of the insects were in non-adult states (eggs, larvae or pupae).Insect species were determined in the Entomology Laboratory, Faculty of Science, Universidad Nacional de Piura.To calculate relative abundance, the number of individuals per species was determined at each sampling.The cumulative relative abundance (%) was calculated and plotted against time.Field and laboratory work was performed in 2004 and 2005.

Isolation of bacteria
Phyllocnistis citrella larvae were collected in Sullana and in Chulucanas.Dead, diseased, and healthy P. citrella larvae were collected directly, by cutting live twigs with their attached foliage from C. aurantifolia trees.Individual larvae were collected without removing them from the tunnels that they had excavated in the leaves.Twigs were kept alive by placing them in a solution of kinetin and gibberellic acid (GA 3 , 200 ppm each) for at least 2 d.Healthy larvae were reserved for biological tests.They were fed on a diet of C. aurantifolia leaves.Collection of insect larvae was constant throughout the project.
Conspicuously diseased larvae were used to for bacteria extraction because they were most likely to contain pathogenic bacteria.Bacterial isolation was performed in the laboratories of the University of Piura and the National University of Piura, Peru.Diseased larvae were sterilized externally by immersion in sodium hypochlorite (0.1%, 1 min) and were then rinsed in sterilized distilled water.Twenty larvae were liquefied and homogenized with a mortar and pestle in 1 mL of distilled water.From the homogenate, 0.1 mL of supernatant was inoculated into nutritive agar (NA) and agar 5% peptone culture media, and incubated at 26ºC (room temperature of the bioassay chambers).
After 24 h incubation, bacterial colonies were isolated and identified by morphological and biochemical tests in the laboratory of the Department of Biology, UNP and the Referential Health Laboratory, LARESA, Piura, Peru.Seventeen different tests were performed on the colonies.The tests were: gram staining, motility, fluorescence, nitrate, oxidase, methyl red, H 2 S, indole, lysine, coagulase, catalase, mannitol, citrate, urea, lactose, production of gas from glucose, and gel at 22ºC.These tests have different specific culture media, indicated in the international protocols.Only in the test for H 2 S the laboratory reported the use of special triple sugar iron agar (Table 1).The results were matched with the responses to these tests of enterobacterial species from the genera studies as reported by Ramírez (1968), Edinger et al. (1985), Aragone et al. (1992), Vivas et al. (2000), Bindu et al. (2003), Traub et al. (1970), and Euzéby (2003).To determine the degree of similarity among bacterial isolates with bacterial species described by the above authors, the number of similar responses between isolated and reference bacteria was divided by the total number of biochemical tests and expressed as a percentage.Pure cultures of each bacterium species were obtained and maintained under standard conditions.Matching was done with reported species of Pseudomonas and Enterobacter, and with the species Serratia marcescens (principal and variants), S. liquefaciens, S. rubida, S. odorifera (two biotypes or variants), S. plymuthica, S. ficaria, S. entomophila and S. fonticola.
To prepare a bacterial inoculum, one colony of each isolated bacterium species was incubated (26ºC, 100 rpm in 5% peptone liquid culture).Absorbance was determined (550 nm) hourly to obtain growth curves of each isolated microorganism.Every hour, 1 mL of pure culture or the suitable dilution was inoculated into solid 5% peptone and incubated at 26ºC.After 24 h, the number of colonies found represented the number of bacteria in 1 mL or CFU.Based on growth curve analysis, bacteria were harvested at the mid-log phase of growth.
By using the culture method and counting CFUs, the bacterial concentration in the different inocula was determined.In the biological test on P. citrella larvae the average concentration of the inocula were 2.2 x 10 6 (Serratia sp.), 4.5 x 10 6 (Pseudomonas sp.), and 2.4 x 10 6 (E.aerogenes) bacteria mL -1 .In the test of acute toxicity in rats, the final bacterial concentration in inoculated wheat was determined; 1 g of inoculated wheat grain was stirred for 5 min in 10 mL of distilled water and 1 mL was used to determinate the CFU.The inoculated wheat had an average concentration of 16 x 10 6 CFU of the three pathogenic species.This was similar to the concentration of used in the pathogenicity test on P. citrella.

Entomopathogenic bacterial activity
The ability of each bacterial species to cause disease was determined.The experimental units were groups of 20 healthy larvae in their leaf mines.Each leaf could have one or more larvae.Each bacterial treatment was replicated three times.
A waxy cuticle covers the photosynthetic tissue or mesophyll of each leaf.Leaf miners carve tunnels as they eat into the mesophyll.They do not eat the cuticle.Cuticles covering the mines were perforated in front of each larva with a sterilized needle to ensure contact between the bacteria and the larvae.A drop of 0.05 mL of liquid inoculum plus a dispersant agent Agridex (1 mL L -1 ) was placed in the leaf tunnel in front of the larvae.When the drop did not totally enter the tunnel in 10 s, excess was absorbed with filter paper.Leaves used as the control were inoculated with sterilized culture medium without bacterial inoculum.Afterwards, larvae were observed in their leaves in a specially designed chamber.Every 24 h, mortality (%) was calculated and the cumulative mortality (%) was plotted against elapsed time since the start of the test.Only bacterium cultures that sickened and killed P. citrella larvae in prior tests were used in the experiment.
The pathogenic effects of the bacterial cultures were tested on individuals of Chrisoperla externa Hagen (Neuroptera) and the convergent lady bird, Hippodamia convergens Guérin-Méneville (Coleoptera).Both prey on insect pests of C. aurantifolia.Chrisoperla externa individuals were obtained from egg samples donated by the National Service of Agrarian Health, SENASA, Peru, and individuals of H. convergens were obtained from field collections.For each bacterial treatment, three groups of 30 individuals of the predator species were maintained in plastic vials (250 mL) covered with a fine cloth.On the first day only, the predators were fed with green bugs (Toxoptera aurantis B. de F.) that had been externally infected by submersion in a liquid culture of each bacterium.Control insects were fed untreated green bugs.Mortality (%) was evaluated as in the previous biological test.
To determinate the pathogenic effect of the bacteria upon C. aurantifolia, liquid inoculums of each bacterium were sprayed (from 20 cm away, for 2 s) on leaves of nursery stock trees (N = 20, three repetitions).Fruit from adult trees were sprayed with the same bacterial suspensions.Groups of 20 limes were used, with three replicates for each treatment.Appearance of symptoms was monitored daily to determine probable phytopathogenic effects.Symptoms on fruits and leaves were expressed as affected area (%) relative to the total surface.

Test of acute mammalian toxicity
In this experiment, the objective was to determine whether the isolated bacteria affected the health of albi-no rats (Mus musculus).Inferences of the bacteria's effect could be made by extension.Three-month-old rats, weighing 18 g on average, were fed wheat grain inoculated with the three bacterial species.To obtain inoculum of each bacterium, 100 mL of culture medium (peptone 5%) was inoculated with a colony of Serratia sp., Enterobacter cloacae, or Pseudomonas sp. and incubated at 26ºC with constant stirring (100 rpm) to obtain an absorbance of 1.5 (550 nm).The cultures were used in the acute toxicity assay.One kg of wheat grain was mixed with 300 mL of each bacterial inocula; the mix was incubated for 24 h at 26ºC and the concentration of each bacterium (CFU) on the wheat was determined.There were 10 rats per group, with three replicates, three bacterial treatments and a control.Rats were fed 2 g of wheat daily.Wheat inoculated with bacteria was used only on the first day.Control rats were fed wheat inoculated with pure liquid culture medium.Afterwards, all rats were fed non-inoculated wheat.The test was conducted for 40 d, during which rat behaviour and mortality were recorded daily.

Bacterial re-isolation
At the end of each test dead insects were sterilized externally and the bacteria were re-isolated using the same procedure as for general bacterial isolation.The re-isolated bacterium species were matched with the bacteria with which the dead insects had been inoculated.

Evaluation
Insect mortality (%) in the biological assays was calculated by subtracting control mortality (without bacteria).Accumulated mortality (%) was plotted against time (h).The mortality dynamic was determined with using a paired t-test for dependent samples with a 95% (P ≤ 0.05) confidence interval.
From October to January, the relative abundance (Fig. 1) of P. citrella was similar to that of the other pests (p = 0.305) and higher than that of the pest control insects (p = 0.009).From February through the evaluation period, the pest-control insects maintained their populations (p = 0.07); while populations of other pests decreased (p = 0.009), and the P. citrella population (p = 0.0006) increased over that of other pests and pestcontrol insects (p = 0.0004).

Bacterial isolation
Bacteria were isolated from larvae infected by the pathogens.Bodies of dead larvae were opaque, dark yellow in colour, and very soft.Diseased larvae were sluggish and generally with amber in colour.The bodies of healthy larvae were slightly yellowish and transparent.They were capable of active movement.
Five bacteria (Table 1) from P. citrella larvae were isolated and identified.The Serratia sp. which was isolated produced gas from glucose and was negative for lysine and urease activity metabolism.Due to differential characteristics, this species was 86% similar to S. pymuthica, 83% and 50% similar to two biotypes of S. marcescens and less similar to other species.Serratia sp. was isolated from insects and was more than 95% similar to S. entomophila, a species isolated from soil and chitinolytic microorganism.The strain obtained here was named only Serratia sp.It is extracted from insects and is very different from Serratia sp associated with human diseases.
Pseudomonas sp. was slightly similar to P. aeruginosa.It grew well under anaerobic conditions but did not reduce nitrate, and did not produce H 2 S gas.On the other hand, it was only 58% similar to P. mendocina and did not induce death in rats.The Enterobacter isolation had 100% identification (all nine tests) with the response patterns of E. aerogenes.Finally, two isolates were identified as Streptococcus sp. and Staphylococcus sp., but were not important because they were not pathogenic on P. citrella larvae.

Pathogenic activity of bacteria
After 24 h the entomopathogenic effect of Serratia sp., Pseudomonas sp., and E. aerogenes under in vitro conditions (Fig. 2A) was similar (p = 0.295).By 48 h, these bacteria had induced average mortalities of 80.2% (Serratia sp.), 70.2% (Pseudomonas sp.), and 71.9% (E.If one compares net maximum mortality among insect species; Serratia sp.induced the highest mortality (between 48 and 72 h).This bacterium was more efficient on P. citrella (80.4% mortality) than on C. externa (43%) and H. convergens (53%).Serratia was 46.5% more efficient on the pest insect than on the biological control insects.
Pseudomonas sp. was more efficient at 72 h on the pest P. citrella (70% mortality) than on C. externa (20%) and H. convergens (10%).The mortality of the most affected controller insect (C.externa) was 71.4% lower than on the pest insect.Enterobacter aerogenes had a similar effect to Pseudomonas, killing 73% of P. citrella and inducing the lowest mortality on C. externa (3%) and H. convergens (30%).The maximum mortality induced by H. convergens was 58.9% lower than on the pest insect.The mortality of P. citrella was statistically similar to that induced by the other two bacterial species.

Acute toxicity of the bacteria to rats
Mortality with Serratia sp (3.3 ± 4%, one rat) was no different from the control at 40 d.The rat died due to natural causes, not from the infection by Serratia sp.With the other bacterial species there were no deaths.Therefore, bacteria pathogenic to P. citrella did not have a pathological effect on rats.The rats, in all treatments, gained weight from 21.5 ± 0.3 g to 26.6 ± 0.2 g.The rats did not show significant difference (p = 0.64) in weight due to the bacteria.

Discussion
On C. aurantifolia, the biocontrol insects C. externa and Hippodamia were present.The P. citrella population was 98% higher than that of other pests, associated with seasonal high relative humidity.The P. citrella population density was highest from February to June; the population of other pests and pest control insects decreased or was constant during the rest of the year.In other work from Peru (Granda et al., 2001;Arce, 2003), the P. citrella population increased exponentially from April to June and reached a maximum in the second week of June and again in the first week of December.This difference may be correlated with the high relative humid-aerogenes), which had statistically similar effects.Average mortality, at 48 h, of P. citrella associated with the three pathogenic bacteria was 74.1%.This was 95% higher (p = 0.0001) than the mortality caused by Staphylococcus sp. and Streptococcus sp.These latter two species were not pathogenic and their effect was similar to that of the control.At the end of the tests, the pathogenic bacteria that were re-isolated from larvae corresponded to the bacteria that were inoculated with.
Serratia sp.induced the highest (p = 0.019) accumulated mortality on C. externa (Fig. 2B): 30% at 24 h and 43% at 72 h.Pseudomonas sp.induced mortality of 14% at 48 h, and a maximum of 20% at 72 h; E. aerogenes induced the lowest mortality (3% at 72 h), which was similar to the control.On H. convergens, Serratia sp.induced the highest mortality rate (Fig. 2C): 40% at 24 h and 53% at 72 h (p = 0.007).Enterobacter aerogenes had an intermediate effect, with maximum mortality of 30% at 72 h; Pseudomonas sp.induced the lowest mortality, 10% at 72 h, similar to the control results.At the end of the tests, the pathogenic bacteria were reisolated from the larvae corresponding to those that had been inoculated.The assayed bacteria did not induce ity of Piura's subtropical summer induced by the effect of the "El Niño" phenomenon.The insects adapt their population dynamics to annual climatic changes.Serratia sp. was the most virulent bacterium.It induced high mortality in H. convergens and in C. externa.It also killed a significant number of P. citrella larvae.On the other hand, S. marcescens is reported to be a nosocomial pathogen (Carrero et al., 1995) and a facultative anaerobe that multiplies quickly in the gut of many insect species, causing septicaemia and death.It is often isolated from diseased and dead insects (Benoit et al., 1990;Rodríguez, 1995;Escobar et al., 2001;Prabakaran et al., 2002;Green et al., 2005).Other species, such as S. entomophila, induce pathologies in pest insects when the bacterium is ingested (Hurst and Jackson, 2002).
The species of Serratia isolated, in this study, had a low matching with S. marcescens, but a high matching with the group S. entomophila; this isolate was innocuous to laboratory rats (Their et al., 1993, Weidenmaier et al., 2004).Hence, the Serratia sp. of this study was used in pest-control experiments on plantations, using traps designed to keep the insects inside (Sepúlveda, unpublished data).Mortality was very high at a low bacterial concentration.As for symptoms of the diseases in insects, sick larvae or those that are killed by bacteriosis become dark-brown or black in colour and appear to be dried and mummified (Bach, 1985;Leucona, 1996) as observed here.Serratia sp. and Pseudomonas sp. have been reported as pathogens of Anastrepha fraterculus, Ceratitis capitata, and Rhynchoporus palmarum (Briceño, 2004); Serratia and other bacteria isolated from fruit flies (A. fraterculus, C. capitata), and R. palmarum induced a crossed effect of a 66.7% of mortality in P. citrella larvae (Campos et al., 2007).
Species of Enterobacter are reported to be normal, or eventual, inhabitants of the gut of healthy insects (Bach, 1985).Natural concentrations of Enterobacter sp. did not induce mortality in C. capitata or A. fraterculus but were pathogenic at high concentrations.The principal symptoms of infection of insects by gram negative bacteria are septicaemia, inhibition of feeding a lack of motility, and death at 24 to 72 h (Briceño, 2004).
Due to their low virulence against C. externa and H. convergens, but high virulence against larvae of P. citrella, it seems that the Pseudomonas sp. and E. aerogenes used in this study would have potential for use in experimental pest control, under controlled conditions, such as the above-mentioned traps.
Enterobacteriaceae are not easy to use for biological control because they are sensitive to dehydration and sunlight, both of which tend to cause variations in bacterial virulence.Further, Pseudomonas and Serratia species include strains with different levels of mammalian pathogenicity (Angus, 1965).However, these species are responsible for natural mortality in insects.This can be taken advantage of, if suitable studies are made and/or the right methods were used (i.e., special traps).For example, S. entomophila and S. proteamaculans are used as effective biological pesticides; they cause amber disease which inhibits insect growth and induces death of C. zealandica (New Zealand grass grub) (Hurst et al., 2000;Hurst and Jackson, 2002).Hippodamia convergens.The bacterial concentration was in the order of 10 6 bacteria mL -1 for all bacteria.Leaves used as controls were inoculated with sterilized culture medium, without bacterial inoculum.
From diseased larvae, it was possible to isolate Serratia sp.Pseudomonas sp. and Enterobacter aerogenes; all of them enterobacteria pathogenic to P. citrella larvae.The isolated bacteria did not have any pathological effect on rats.This could be important in deciding to use these bacteria in programs or systems for pest control of P. citrella.Serratia sp. was the most virulent against the P. citrella predator insects, C. externa and H. convergens.The other bacteria were almost harmless to the predator insects, but caused death of the pest.This is an important consideration because the bacterial concentration can be determined to achieve maximum pest death and minimum death of the biocontrol species in pest control programs.This technology needs further development to be implemented at all levels of production.

Figure 2 .
Figure 2. Entomopathogenic effect of isolated bacterial species on: (A) Phyllocnistis citrella larvae.(B) Chrisoperla externa.(C)Hippodamia convergens.The bacterial concentration was in the order of 10 6 bacteria mL -1 for all bacteria.Leaves used as controls were inoculated with sterilized culture medium, without bacterial inoculum.

Table 1 .
Characteristics of bacteria isolated from Phyllocnistis citrella larvae; +: positive reaction, -: negative reaction, (): low reaction, a: using triple sugar iron agar any damage or symptoms on leaves or fruit of Citrus aurantifolia.