Kinnow affected by greening (photo courtesy of Mr. Muhammad Fakhar-ud-Din Razi)

Citrus is the major fruit crop in Pakistan, where Kinnow mandarin is presently the dominate cultivar, accounting for 80 percent of the acreage. Most graft-transmissible pathogens (GTP) of citrus present in Pakistan are transmitted to new plants only by using infected nursery materials for propagation and do not have insect vectors. These GTP may be effectively controlled by using pathogen-tested (pathogen-free) propagating source materials. However, several important GTP have insect vectors, such as Huanglongbing (HLB) or citrus greening and citrus tristeza virus (CTV), and these pathogens result in a short economic life of production groves and reduced tree vigor, yield, and fruit quality. HLB, caused by a phloem-inhabiting bacterium, is the most devastating disease of citrus and is spread by psyllid insects. HLB is endemic in Asia and was reported in Brazil in 2004 and in Florida in 2005. In addition to reducing yield and tree vigor, affected fruit has a bitter taste, making it unmarketable even for juice. CTV is spread by several species of aphids, and severe stem pitting strains of CTV impact both trees and fruit.

The primary objectives of this project are to (1) develop an infrastructure to treat local germplasm in Pakistan to ensure that it is free of greening; (2) evaluate citrus and related plants for greening resistance and tolerance; and (3) apply real-time PCR assays for detection of Huanglongbing (HLB) virus and citrus tristeza virus to study how the pathogens spread under different field conditions. In Pakistan, three groves were selected for monitoring in each of four major citrus growing regions, and five trees were tagged within each grove, with each being monitored weekly for psylla populations. All trees were scored for symptomatic presence of HLB virus, with a 9% incidence of HLB symptoms at the beginning of the observations. By the end of the first quarter of 2009, the U.S. partners had shipped seeds from 92 citrus varieties and related plants, and although about 5% of the seed samples were overly dry upon arrival and had to be recollected for reshipment, the rest were sown in a protected greenhouse in Faisalabad. Now that the seedlings are at the 6-12 leaf stage, they are ready to be moved from the greenhouse to the field. Five plants of each variety will be planted into the field in a replicated, random planting where they will be naturally exposed to HLB spread by the psyllid vector, Diaphorina citri. 

A suction psyllid trap similar to the one shipped to UAF (photo courtesy of Dr. Richard Lee)

A 2-meter-tall suction trap has also been shipped to the University of Faisalabad and was installed in the summer of 2009, and UAF upgraded its labs with a gel electrophoresis system, PCR machine, genetic analyzer, and several other pieces of equipment funded through this project. Arrangements have made to ship some of the psyllids from Faisalabad to Riverside, where they will be tested for presence of the bacterium associated with HLB using real time PCR, with similar tests to be conducted at UAF as well. Meanwhile, the postdoctoral researcher employed on the project in Riverside has been sequencing a 1.6 kb fragment of the nuclear gene, malate dehydrogenase, and is using the single nucleotide polymorphisms to determine the phylogenetic relationships among Citrus species and the citrus relatives. Some preliminary results were presented at the annual meeting of the American Phytopathological Society in July 2009 and at the Plant and Animal Genome XVIII Conference in San Diego in January 2010.

According to the US co-principal investigator Dr. Richard Lee, a more rapid method is needed to eliminate HLB from citrus germplasm so that the germplasm can be used as clean sources of buds for the propagation of healthy plants. Conventional thermotherapy of citrus has not been efficient and while shoot tip grafting works well, it is time consuming and requires a high level of expertise by the technician. Experiments have begun to study the use of antibiotics and/or heat therapy of budwood as a way to quickly eliminate HLB from budwood. In Riverside, theCandidatus Liberibacter associated with tomato psyllid yellows is being utilized to determine the effectiveness of various antibiotics to eliminate this pathogen in tomato, and citrus stubborn, a phloem-limited spiroplasma disease endemic to California, is being used as a model system to determine the tolerance of citrus to the antibiotics being tested and tolerance to different heat regimes and effectiveness in eliminating stubborn. The results from these preliminary trials have been transferred to cooperators in Pakistan for trials with citrus infected with HLB.

The devastating effects of greening are clearly visible in the oranges at right, compared with healthy fruit at left (photo courtesy of Mr. Muhammad Fakhar-ud-Din Razi)

In 2010 the partners on this project are looking forward to the six-month visit of a Pakistani PhD student to Riverside beginning in the first quarter of the year. He will receive training in the molecular characterization of HLB and detection of HLB in psyllids and plants. A second student from Pakistan will visit Riverside later in the year to receive training on shoot tip grafting and thermotherapy of germplasm, and he will work in the research area of antibiotic/temperature treatments to eliminate greening from budwood and development of multiplex real time PCR for detection of multiple prokaryotic pathogens in citrus. Dr. Lee will visit Pakistan in 2010 if travel restrictions are lifted, but meanwhile research will continue in Riverside and Pakistan on the use of antibiotics and variations of temperature treatments of budwood to eliminate HLB. Further details on project results and upcoming activities may be found in the annual progress report for 2009 linked near the top of this page.