An Environmentally-Friendly Alternative for Control of the Citrus Nematode in Arizona

2007 Annual Report for GW07-007

Project Type: Graduate Student
Funds awarded in 2007: $19,746.00
Projected End Date: 12/31/2008
Grant Recipient: University of Arizona
Region: Western
State: Arizona
Graduate Student:
Major Professor:
Dr. Patricia Stock
Entomology-University of Arizona

An Environmentally-Friendly Alternative for Control of the Citrus Nematode in Arizona


The citrus nematode, Tylenchulus semipenetrans, has been reported to affect 90% of Arizona’s citrus. At present not many options exist for control of this pest. Most chemical nematicides used in the past are no longer available. This situation urges the search for new control alternatives. Biocontrol agents such as entomopathogenic nematodes (EPN) are one potential option. EPN have shown to control some plant–parasitic nematode species. In this project we propose to assess EPN for control of T. semipenetrans. If proven effective, EPN will provide an alternative option to chemical-control for implemention in Arizona and other citrus-producing regions in the country.

Objectives/Performance Targets

The overall goal of this study is to assess the effect of commercially available and Arizona-native entomopathogenic nematodes (EPN) as an alternative tool for control of the citrus nematode, T. semipenetrans.
Specific objectives are:

1. To conduct lab experiments to determine the best EPN “species-match” for control of the citrus nematode considering two commercially available EPN S. riobrave (Biovector) and H. bacteriophora (Nemasys) and two Arizona-native EPN (Steinernema sp. ML18 isolate, and Heterorhabditis sp., CH35 isolate).
2. To conduct two field trials in Yuma, AZ considering the two best-performing EPN isolates tested.


During 2007 we focused on Objective 1: “To assess the best EPN “species-match” (under laboratory conditions) for control of the citrus nematode considering S. riobrave (Biovector®) and H. bacteriophora (Nemasys®)”. For this purpose, rough lemon seedlings were considered for all experiments. Briefly, seeds were individually planted in 4 cm diameter cone-tainers® (Figure 1), previously filled with sterilized sand (Figure 1A).
T. semipenetrans J2s were isolated from tangelo roots from the UA Mesa Agricultural Station in Yuma, AZ. Sampling consisted in extracting citrus feeder-roots from orchards at this station or from other citrus nematode-infested private orchards in the area. Roots were collected at least 2 weeks prior to each experiment and J2s were extracted using a modified mist extractor (Barker, 1985) Briefly, feeder roots (approximately 25-50 g) were placed on Baermann funnels, inside a mist extraction chamber. A mist was applied to the sol and root samples inside the chamber at 5 min intervals, moistening the roots and allowing nematodes to migrate into the water. After 4 days, the water collected from each funnel was retrieved and the total number of T. semipenetrans J2s was accounted, considering volumetric dilutions.

Entomopathogenic nematodes, Steinernema riobrave (Biovector®) and H. bacteriophora (Nemasys®) (Figure 1B) were reared in vivo using G. mellonella larvae (Kaya and Stock, 1997). Infective juveniles (IJs) of these nematodes were harvested from White traps (White, 1927) and stored in water at 15C prior to application, for a period no longer than 2 weeks. Two EPN-inoculation approaches were considered for assessing interactions between this nematode and T. semipenetrans. The first approach was the inoculation of third-stage infective juveniles (J3) as a water suspension (Figure 1D). The second approach was the application of EPN-infected G. mellonella cadavers directly into the soil (Figure 1E). Justification for this approach was based on studies conducted by Shapiro et. al (2003). Their studied indicated that EPN application of infected insect cadavers tended to be more efficacious that aqueous applications because the cadaver applications in the soil kept IJs of entomopathogenic nematodes under less physiological stress.
Two application times were considered: 1) simultaneous application: this is both citrus nematode and EPN applied into the soil at the same time, and 2) after application: this is establishment of citrus nematodes first (i.e. for an 8-week period) (Figure 1C), and inoculation of EPN after this 8-week period. Seedlings were allowed to grow for a two-month. Once the citrus seedlings reached the desirable root length, an aqueous suspension of 12,000 T. semipenetrans J2s was added to each cone-tainers®.
EPN were applied as either a liquid or in the insect-infected G. mellonella cadaver alternative for the simultaneous inoculation time. A concentration of 250 IJ/cm (i.e. total of 1,000 IJs/cone-tainers®) was considered for the aqueous EPN suspension. For the EPN-infected G. mellonella cadaver, 1 single insect larva was buried in the soil at 3 cm depth. G. mellonella cadavers were infected with 100 IJ/Galleria 48 hr prior to their inoculation in cone-tainers® s.
Controls consisted of nematode-free plants; this is healthy citrus seedlings that received an equal amount of water to that applied for the nematode aqueous inoculate. Additional controls consisted of a) citrus seedlings inoculated with T. semipenetrans only, b) citrus seedlings inoculated with EPN liquid suspension only, c) citrus seedlings inoculated with EPN-infected insect cadaver only. A completely randomized experimental design was used. Each treatment was replicated 12 times.
EPN were allowed to interact with T. semipenetrans for an 8-week period. At that time the citrus seedlings were removed for assessment of T. semipenetrans establishment in the roots and to account for egg production. T. semipenetrans eggs were extracted from the seedlings roots using a 20% bleach solution according to procedures described by (Hussey and Barker, 1972). Eggs were then washed through two mesh screens (80 and 500 mesh screens) and rinsed into a beaker with dH2O.
Eggs were counted under a dissecting microscope at a magnification of 50X. Soil from the cone-tainers® s was also examined to account for T. semipenetrans J2 and for S. riobrave J3s present in each sample. Each soil sample was saved from each plant separately and put up on the mist extractor (Barker, 1985) to account for nematode presence. Samples collected from the mist extractor were poured over a 500 micron mesh screen and rinsed with deionized H2O to collect nematodes. Both EPN IJs and T. semipenetrans J2s were counted under a dissecting microscope at a magnification of 50X. Fresh root weight was also determined for all groups.
Data was subjected to analysis of variance using Sigma Stat. Experiments were conducted three times and analysis were combined and run as a block. Treatment means were compared and P-values were determined.

Results from these experiments showed the EPN S. riobrave can cause reduction in egg production and J2 population in the soil of the citrus nematode with either application method (i.e. aqueous suspension and infected cadaver) and application time (i.e. simultaneous and after) (Figures 2,3). Contrarily, H. bacteriophora does not seem to suppress the citrus nematode. According to our results only a reduction in egg production and J2 population, was observed with the infected cadaver simultaneous application (Figures 4,5). These results suggest that at least the EPN S. riobrave can reduce infection of T. semipenetrans to citrus roots.

NOTE: Figures 1-5 are included in CD and hard copies of this report.

b. Outreach activities
During this period, preliminary results of the ongoing research were presented at the Joint Meeting of the AMERICAN PHYTOPATHOLOGY SOCIETY and the SOCIETY OF NEMATOLOGISTS, San Diego, CA, July 29-August 2, 2007.

c. Future activities
During the next funding period we will finalize laboratory testing of two Arizona-native EPN species ML-18 (Steinernema sp.) and Ch-35 (Heterorhabditis sp.). This research has already been initiated and will finalize in June 2008. During the second half of 2008 we will focus on the field evaluation of the most effective EPN species/formulation. This field trials will be conducted in Yuma Arizona.

Literature cited

Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K. 1988. Current Protocols in Molecular Biology. Wiley Interscience, Inc.

Barker, K.R. 1985. Nematode extraction and bioassays. In: An Advanced Treatise on Meloidogyne Vol II Methods. K.R. Barker, C.C. Carter, J.N Sasser (eds.) North Carolina State University, Raleigh, North Carolina Pp 19-65.

Hussey, R.S. and Barker, K.R. 1973. A comparison of methods of collecting inocula of Meloidogyne spp., including a new technique. Plant Disease Report 57, 1025-1028.

Kaya, H.K. and Stock, S.P. 1997. Techniques in insect nematology. In: Lacey, L. (ed.), Manual of Techniques in Insect Pathology. Academic Press, New York. Pp. 281-322.

Shapiro-Ilan, D.I., Lewis, E.E., and Tedders, W. L. 2003. Superior efficacy observed in entomopathogenic nematodes applied in infected-host cadavers compared with application in aqueous suspension. J. Invert. Path. 83: 270-272.

White, G. F. 1927. A method for obtaining infective nematode larvae from cultures. Science 66: 302-303.

Impacts and Contributions/Outcomes

Sustainable IPM in the 21st century will rely increasingly on alternative strategies for pest management that are environmentally friendly and reduce the amount of human contact to chemical pesticides. One of the most promising choices to help minimize usage of chemical pesticides is the implementation of EPN. Many species are currently employed as biological control agents of insect pests and plant-parasitic nematodes. With EPN, we have the opportunity to develop and implement technology that will significantly reduce the transmission of disease, protect biodiversity, enhance water quality, preserve the environment and improve food safety and affordability. This aspect is of crucial need in desert ecosystems like the one in southwestern US.
Through this WSARE-funded research we are assessing EPN as an alternative tool for control of the citrus nematode, T. semipenetrans. Preliminary results from laboratory assays indicate that at least one EPN species, S. riobrave is able to reduce iJ2 populations and egg production of established females of the citrus nematode in roots of rough lemon seedlings. If these results are successful in field tails planned for summer 2008, the consideration of EPN for control of this plant-parasite will provide an environmentally safe alternative to traditional chemical control that could be implemented not only in Arizona, but in other citrus-producing regions in the country. This aspect intimately relates to WSARE goals 1 and 5, which promote profitable sustainable farming methods to help maintain and enhance the quality of the soil, and conserve natural resources and wildlife.
In addition to this, the effective control of this citrus pathogen will eventually encourage growers to continue growing citrus in Arizona, therefore increasing crop diversification in the Southwest. This aspect relates to WSARE goals 2 and 4. Moreover, this ongoing research also correlates to WSARE goal 3, which advocates the adoption of methods and agricultural practices that reduce potential risks to human health and the environment caused by pests themselves or by the use of pest management practices. Furthermore, the development an environmentally safe and effective biocontrol agent (i.e. EPN) could greatly improve crop and yield quality, therefore enhancing the quality of life of citrus growers in this region. This aspect relates to WSARE goal 2, which promotes the enhancement of quality of life of farmers and ranchers and ensures the viability of rural communities by increasing income and employment in agricultural and rural communities.


Chan Maketon
Undergraduate Research Assistant
Dept. Entomology, Univ. Arizona
Forbes 410
1140 E. South Campus Dr.
Tucson, AZ 85721
Office Phone: 5206211317
Rousel Orozco
Reserach Assistant
Dept. Entomology. University of Arizona
Forbes 410
1140 E. South campus Dr.
Tucson, AZ 85721
Office Phone: 5206211317
Yolanda vega
Research Associate
University of Arizona
Dept. Entomology
Forbes 410. 1140 e. South campus Dr.
Office Phone: 5206261317
Joanna Gress
Graduate Student
Dept. Plant Sciences, Univ. Arizona
1140 E South Campus Dr.
Tucson, AZ 85721
Office Phone: 5206211317