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- Widmer, Timothy
- Norrington, Bryan
- 2023
- 2022
- Crop Protection & Quarantine (304)

Research Project: Exploration for Natural Enemies in China for Citrus psyllid in the U.S. **Location:
                            Office of International Research Engagement and Cooperation** 2022 Annual Report **Objectives**
                                

Objective 1: Discover, identify and initiate the development of new biological control agents for citrus psyllid [NP304, C3, PS 3A]
Sub-objective 1.A: Setup malaise traps to collect insects in representative geographical locations in Yunnan and Guangdong provinces in China.
Sub-objective 1.B: Apply DNA metabarcoding technology for high throughput analysis of the collected samples, establish corresponding DNA barcode database.
Sub-objective 1.C: Use sentinel and wild egg masses/ nymphs of citrus psyllid to examine parasitism and the species composition of natural enemies.
Sub-objective 1.D: Field survey and collecting predators observed to be actively feeding on ACP, and parasitoids emerging from ACP.

Objective 2: Evaluate the biocontrol potential of biocontrol agents of citrus psyllid under laboratory and field conditions and develop the technology of mass rearing. [NP304, C3, PS 3B 3C]
Sub-objective 2.A: Evaluate the biocontrol potential of biocontrol agents of citrus psyllid under laboratory and field conditions.
Suitability and preference of predators.
Sub-objective 2.B: Develop the technology for mass rearing of some biocontrol agents. **Approach**
                                

Survey for, collect and identify natural enemies of agricultural and landscape 
pests. Make field collections, establish laboratory colonies when needed, and ship candidate agents to the U.S. for further evaluation. Conduct preliminary host specificity tests on promising parasites, predators, and beneficial microorganism. Collect, colonize, and ship to the U.S., candidates found to be safe. Cooperate with USDA and State laboratories in future collections for shipment and evaluation for release. Collect natural enemies attacking pests at differing host densities and climatic zones. All natural enemies selected for shipment will be sent directly to State or Federal quarantine facilities in the U.S. for further evaluation. Both Chinese and American scientists share the same right of obtaining biological control agents collected from the two countries. Assist in obtaining the necessary regulatory agency permissions for exporting live material from China. **Progress Report**
                                

3a. Under Objective 1, Sub-Objective 1A- Setting up malaise traps for insect sample collection. local collaborators and Sino-ABCL research scientists have set up in the main citrus growing areas of Yunan and Guangdong provinces in China.  Guangdong and Yunnan are the major citrus-producing provinces in China. Many wild and semi-wild citrus species and related genera are found in Yunnan. There is rich insect biodiversity in Guangdong and Yunnan, where humid climatic conditions predominate.  Malaise traps have been set up in main citrus growing areas of Yunnan and Guangdong provinces in China. Citrus orchards without a history of pesticide use will be selected as the experimental sites. 

3b. Under Objective 1, Sub-Objective 1B- Insect sample DNA metabarcoding data analysis. Sino-ABCL started sequencing and DNA metabarcoding the samples collected from citrus orchards. Widespread interest in metabarcoding has resulted in data proliferation and the development of computational tools to aid data analysis. One such tool, mBRAVE, the Multiplex Barcode Research and Visualization Environment, is a data storage and analytics platform with standardized pipelines and a sophisticated web interface designed to transform raw HTS data into biological insights. mBRAVE integrates common analytical methods and links to the BOLD System for access to reference datasets. The current study analyzed the composition of insect communities in citrus orchards using DNA metabarcoding followed by data analysis on BOLD and mBRAVE. The species revealed by BIN matches on BOLD were then searched in the literature to allow their classification into pest, parasitoid, predator, or pollinator.

3c. Under Objective 1, Sub-Objective 1C- Sub-objective 1.C: Use sentinel and wild egg masses/ nymphs of citrus psyllid to examine parasitism and the species composition of natural enemies. We have established citrus psyllid populations in the laboratory by screening the host plants, that can produce enough sentinel egg masses of ACP for the field experiments. Hosts of ACP in China include up to 27 species within seven genera of Rutaceae. Murraya exotica (Rutaceae) is commonly grown in citrus production areas and has been shown to be good hosts of the Asian citrus psyllid. Therefore, we used M. exotica and citrus as the host plants for rearing the citrus psyllid in our laboratory. The population of citrus psyllid has been established indoors.

3d. Under Objective 1, Sub-objective 1.D: Field survey and collecting predators observed to be actively feeding on ACP, and parasitoids emerging from ACP and Sub-objective 2.A: Evaluate the biocontrol potential of biocontrol agents of citrus psyllid under laboratory and field conditions. Tamarixia radiata (Hymenoptera: Eulophidae) is an ectoparasitoid of the Asian citrus psyllid. Interest in T. radiata for biological control of psyllid has grown in response to continued spread of HLB, and the evident searching and colonization capabilities of the parasitoid. We collected T. radiata in 5th instar ACP mummies and collected the specimens for morphological research and evaluation of biocontrol potential. In addition, we also collected predators including lady beetle, lacewing and Arma chinensis for the research of predation and evaluation of biocontrol potential.

3e. Under Objective 2, Sub-objective 2.B: Develop the technology for mass rearing of some biocontrol agents, we continue to develop the technology of mass rearing of biological agents.  Large numbers of high-quality biological control agents are fundamental prerequisites to successful integration of augmentative biological control and thus, to the success of mass rearing programs. Several predator colonies are maintained in Sino-ABCL, including A. chinensis, lacewing, and lady beetle. We developed artificial diets for these predators and improved the rearing apparatus and environmental conditions accordingly. Studies on evaluation of the quality indicators for the mass rearing of biological agents were also conducted. **Accomplishments**
                                

1. Using DNA metabarcoding to assess insect diversity in citrus orchards. These bulk samples were metabarcoded, and sequences were curated and analyzed using two cloud-based data storage and analytical platforms, the Barcode of Life Data System (BOLD) and Multiplex Barcode Research And Visualization Environment (mBRAVE). Species which received a species match on BOLD were placed in one of four categories based on this assignment: pest, parasitoid, predator, or pollinator. As this study provides the first baseline data on insect biodiversity in Chinese citrus plantations, it is a valuable resource for research in a broad range of areas such as pest management and monitoring beneficial insects in citrus gardens. 2. Heat shock protein superfamily in Arma chinensis and the responses to temperature stress. Temperature is an important environmental factor in agriculture, affecting individual organisms and the entire farmland ecosystem. Global warming has become more tangible, which may negatively affect pest biological control due to the generally weak thermal tolerance of natural enemies. Arma chinensis (Hemiptera: Pentatomidae) play an important role in the natural control of citrus orchard pests. However, the effect of thermal stress on the predator remains poorly understood. Firstly, in our current research, we identified 57 hsp genes, including five hsp90, 13 hsp70, eight hsp60 and 31 shsp (small heat shock protein) genes. The results about the hsp gene expression profile under elevated temperatures showed at least three hsp gene families are in response to temperature stress. The predatory ability of A. chinensis was negatively impacted by high temperatures, with a significantly longer handling time at elevated temperatures. Even so, we also found A. chinensis was more heat tolerant to its prey pests and exhibited better development, survival, and reproduction features. Our results highlighted the molecular evolutionary properties and the response mechanism to temperature assaults of hsp in A. chiensis.


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