Final Report for GW07-007
The citrus nematode, Tylenchulus semipenetrans, has been reported to affect 90% of Arizona’s citrus. At present few 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 been 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 to chemical-control for implemention in Arizona and other citrus-producing regions in the country.
The citrus nematode, Tylenchulus semipenetrans (Nematoda: Tylenchulidae), is one of the most debilitating citrus pests worldwide (Duncan and Cohn, 1990; Verdejo-Lucas and Kaplan, 2002). This nematode is an obligate parasite that reproduces only on the living roots of host plants. The nematode female becomes semi-endoparasites and sedentary by burrowing its anterior end deep inside the root cortex while the posterior end remains outside in the soil. It establishes feeding sites within the root cortex composed of nurse cells that surround the female nematode head. The posterior portion of the adult female protrudes from the root and is surrounded by a gelatinous matrix in which eggs are deposited (Cohn, 1965).
In Arizona T. semipenetrans was first discovered in 1926 and, since then, citrus growers have faced a continual battle with this citrus pest. Ninety percent of the citrus in the state has been reported to be affected by this nematode (Olsen et al., 2000). Although this nematode does not kill the trees, it significantly reduces tree vigor, growth and grove productivity (Duncan and Cohn, 1990). For this reason, the disease is often referred to as a “slow-decline.” Above-ground symptoms can include leaf yellowing, sparse foliage, small and non-uniform fruit and defoliated upper branches. Yield losses caused by T. semipenetrans are estimated at about 10% worldwide or $77 million per year (Verdejo-Lucas and Kaplan, 2002). At present very few alternatives are available for control of this nematode. Most chemical nematicides previously considered for control of this nematode (i.e. aldicarb, fenamiphos, DBCP) have been or soon will be removed from the market due to their known toxicity and detrimental health effects to humans, wildlife and soil and ground water contamination (McClure and Schmitt, 1996; Anonymous, 2002). This situation and the increasing awareness of environmental and human health concerns associated with chemical nematicides urges the search for new alternatives of environmentally friendly products for management of this nematode. In this respect, one of the choices for substitution of chemical nematicides is the consideration of biological control agents such as entomopathogenic nematodes (hereafter referred as to EPN). EPN provide an environmentally safe and economically reasonable alternative for a wide range of arthropod pests and plant-pathogens (Tanada and Kaya, 1993). With respect to plant-parasitic nematodes, several greenhouse experiments and field trials have shown the ability of EPN to reduce plant–parasitic nematode penetration and egg production (Lewis et al., 2001; Jagdale et al., 2002; Pérez and Lewis, 2004). EPN are already available as commercial products and can directly be used for nematode control thus reducing cost and time of product development. Preliminary laboratory tests conducted at U of A with Steinernema riobrave show good promise of this EPN to control citrus nematode. Two application types were considered: nematode aqueous suspension and EPN-infected cadavers. Our results indicate that S. riobrave-infested cadavers in the soil significantly reduce egg production and penetration in rough lemon seedlings (Gress & Stock, 2005. Abstract- 10th European IOBC Meeting)
Over the past decades, the prevailing use of chemical pesticides has generated several problems including insecticide resistance, outbreaks of secondary pests, safety risks for humans and domestic animals, contamination of ground water, decrease in biodiversity among other environmental concerns (Lacey et al., 2001). These problems and sustainability programs based mainly on conventional insecticides have stimulated increased interest in the development and implementation of cost-effective, environmentally safe alternatives to chemical pesticides for arthropod and plant-pathogens control. 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 in row and glasshouse crops, orchards, ornamentals, range, turf and lawn, store products and forestry (Lacey et al., 2001). 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 U.S.
In this project we assessed EPN as an alternative tool for control of the citrus nematode, T. semipenetrans. Use of EPN as a substitute for chemical pesticides for control of agricultural pests is not a novel idea, but no EPN have yet been tested against this specific plant-parasitic nematode. In order to effectively implement EPN for the control of this plant-parasitic nematode, some basic research needs to be established. In this respect, data generated from this study will assist in the subsequent development and application of EPN as an alternative to citrus pest control practices. If proven effective, 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 Western SARE 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 Western SARE goals 2 and 4.
Moreover, the proposed research also correlates to Western SARE 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 is addresses Western SARE goal 2, which promotes the enhancement of quality of life of farmers and ranchers and ensure the viability of rural communities by increasing income and employment in agricultural and rural communities.
Objective 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). Objective 2. To conduct field trial(s) in Yuma, AZ considering the two best-performing EPN isolates tested.
Objective 1. Assays will be conducted on 2-months-old rough lemon seedlings reared in cone-tainers, in growth chambers at 25 ºC and 30% humidity. Two EPN application times will be considered: a) SIMULTANEOUS: EPN simultaneously applied with citrus nematode, b) AFTER: EPN applied after citrus nematode establishment in the citrus seedlings. For each of these application times the following treatments will be considered: i) EPN aqueous suspensions (2 concentrations: 1,000 and 10,000 IJ/ seedling); ii) EPN-infected cadavers (with an exposure rate of 100 IJ/wax moth). This second approach is based on a study conducted by Shapiro et al (2003). Their study indicated that EPN application with infected cadavers tends to be more efficacious than aqueous applications because the cadaver applications were under less physiological stress (i.e. osmotic stress). Controls will consist of: i) nematode-free citrus seedlings, water added only; ii) T. semipenetrans-infested seedlings; iii) citrus seedlings with EPN aqueous suspension only; iv) citrus seedlings with EPN-infected cadavers only, and water added. T. semipenetrans-infected seedlings will be inoculated at a concentration of 12,000 J2s/ seedling. A completely randomized design with 12 cone-tainers; (citrus seedlings) per treatment for each application time will be considered. Eight weeks after EPN inoculation, citrus seedlings will be removed for assessment of T. semipenetrans establishment in the roots and egg production. Data will be subjected to analysis of variance using StigmaStat analysis software. Experiments will be repeated three times. The EPN that performs best in controlling the citrus nematode in the growth chamber trials will be considered for field trials.
Objective 2. Two field trials were considered. Tests were conducted in late June and mid-August September 2007. Plots were arranged in two row beds in a randomized design with 3 experimental treatments and 10 replicates. An in-row variable tree buffer was established in each trial to prevent treatment interference. Different EPN doses were considered to assess the effect of this nematode on citrus nematode penetration, egg production and citrus root growth.
EPN doses considered were: 1) low: 54 nematodes/cm2, 2) medium: 108 nematodes/cm2, and 3) high: 532 nematodes/cm2. Nematode suspensions were premixed in 2.5 gal of water and each mixture was added to the tank of a CO2 pressurized hand sprayer, which has flat fan nozzles. Additionally, EPN-infected Galleria mellonella larvae cadavers (infected 2 days prior application day) were buried in the soil surrounding the drip-line of the tree following procedures described by Perez et al., 2003). Controls considered water application only.
Sampling of citrus nematodes was conducted in both orchards prior to and after (6-8 weeks after application) EPN treatments. Ten soil samples of approx. 100 g were taken randomly from the orchard. Samples were processed in a mist extraction chamber to recover and account for Tylenchulus nematodes. Data recovered from both trials were subjected to analysis of variance using SAS statistical software package.
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).
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.
Objective 2: To conduct field trial(s) in Yuma, AZ considering the two best-performing EPN isolates tested.
Field trials showed a reduction on citrus nematode populations (J2 and males) when considering the infected cadaver alternative. However, no significant reduction of T. semipenetrans populations was achieved when considering the aqueous suspension (Biovector formulation).
Educational & Outreach Activities
-Stock, S. P. 2007 Nematodes and Agriculture in the Southwest: The Good, the Bad and the Ugly. Yuma Ag Summit, Yuma, Arizona, March 2007.
- Gress, J. C. and Stock, S. P. 2007. Nematodes against nematodes: assessing entomopathogenic nematodes (Steinernematidae, Heterorhabditidae) for the control of the citrus nematode Tylenchulus semipenetrans. American Phytopathology Society and Society of Nematologists Joint Meeting, San Diego, CA, July 29-August 2, 2007.
-Stock, S. P. 2008. Insect-Pathogenic Nematodes and their Potential for Citrus Pest Control in the Sonoran Desert. International Congress of Entomology, Durban South Africa, July 6-12, 2008.
-Stock, S. P. and Maketon, C. Assessment of entomopathogenic nematodes (Steinernematidae, Heterorhabditidae for control of the citrus nematode Tylenchulus semipenetrans (Tylenchida: Tylenchulidae) under laboratory conditions. In preparation (to be submitted to Journal of Invertebrate Pathology).
According to data generated through this project we consider entomopathogenic nematodes such as Steinernema riobrave may be utilized as a biological control alternative of the citrus nematode T. semipenetrans. If optimization of application in citrus orchards is achieved we speculate consideration of entomopathogenic nematodes for citrus nematode control may be feasible.
Ms. Joanna Gress graduated in August 2007 with a Masters degree in Plant Sciences.
Not applicable to our project (at least not for this preliminary study).
Not applicable to this project (at least not for this preliminary stage of our research).
Areas needing additional study
According to data generated through this project we believe entomopathogenic nematodes such as Steinernema riobrave may be utilized as a biological control alternative of the citrus nematode T. semipenetrans. One critical aspect that requires further investigations is the optimization of the application of entomopathogenic nematodes. It seems that the cadaver application would be a better alternative compared with the aqueous suspension. Research on appropriate cadaver formulation and applications needs to be further investigated.
Our results indicate that entomopathogenic nematodes have an antagonistic effect on the development of the citrus nematodes in the citrus roots. The mechanisms involved in this antagonism are yet not known. Future research should be focused on unraveling the mechanisms involved in this antagonism.