1a. Objectives (from AD-416)
Determine genetic diversity of tristeza and stubborn disease agents in California; 2)Characterize tristeza and stubborn biologically by graft and vector passage; and 3)Examine patterns of spatial and temporal spread of tristeza and stubborn.
1b. Approach (from AD-416)
Isolate, identify, and characterize field strains of tristeza and stubborn in California with respect to phenotype, vector transmissibility, serology, molecular structure and phylogeny, and epidemiology. This will be accomplished by using a combination of the following: enzyme-linked immunosorbent assay (ELISA); reverse transcription (RT) polymerase chain reaction (PCR); conventional PCR; real time PCR; transmission electron microscopy; dark field microscopy; bacterial culturing in cell-free media; cloning; sequencing; spectrophotometry; electrophoresis; insect transmission; geostatistics; and ARC-GIS. Replaces 5302-22000-006-00D.
3. Progress Report
Cross protection tests against Citrus tristeza virus (CTV) showed that a CTV strain which protected against seedling yellows expression did not ameoliorate stem pitting expression. A combination of strains will be necessary to protect citrus against seedling yellows and stem pitting. Aphid transmission and sequencing of aphid-transmitted subisolates indicated the seedling yellows protecting isolate was a complex of three different CTV strains. Individual aphid transmitted sub-isolates failed to prevent seedling yellows but, when reinoculated as a mixture, regained cross protection. Small interfering viral RNAs and host micro RNA profiles were associated with seedling yellows symptom amelioration. Tests are underway to validate these results which, if true, could be used to develop a biomarker for cross protection. In collaboration with UC’s Lindcove Field & Extension Center, Exeter, CA, spread of potentially virulent strains of CTV were monitored by serology and real-time polymerase chain reaction (PCR) in a one-square-mile area surrounding the LREC. Since 2009, this area has been treated biannually with insecticides for aphid control as part of a grower-funded regional pilot program to limit spread of CTV and trees tested for infection by virulent CTV strains. In 2010, fewer than 20 trees in this area tested positive to MCA13, a CTV strain discriminating monoclonal antibody. Among these isolates, the T3 strain was found by molecular assay in several trees and all trees with MCA13 positive isolates were eradicated. In the spring 2011, only 11 of 10,850 trees tested (0.1%) at the LREC were found infected by CTV; none by the virulent T3 strain. On average over the three previous years, CTV was detected in 60 trees per year. These results suggest vector control may significantly mitigate the spread of CTV. Analyses of a three-year study of incidence of Spiroplasma citri-infected trees in two mature citrus orchards in central CA indicated that disease distribution was due to primary spread with an absence of secondary spread. Few newly infected trees were detected in each grove despite differences large differences in incidence among plots, ranging from 4 to 22%. In contrast, a high rate of infection was found in another plot of young citrus next to row crops rotated between carrots, tomato, onion and parsley. In this case, incidence of S. citri-infected trees decreased with distance from row crops. Leafhopper vectors in parsley were collected and tested positive for Spiroplasma citri, causal agent of citrus stubborn disease. These data suggest S. citri-infected trees were not a good source of inoculum for vector spread of the pathogen. Natural spread of S. citri was, thus, dependent on a crop or weeds which supported inoculative leafhopper vectors that transiently feed on citrus during movement from one row crop to the next.
1. Small interfering RNAs and citrus micro RNAs in Citrus tristeza virus-infected plants. The mechanism of cross protection is unknown. ARS researchers at Parlier, CA, in collaboration with Consiglio Nazionale delle Richerche (CNR)scientists in Bari, Italy, determined sequences of small RNAs from sour orange citrus with asymptomatic and severe seedling yellows to determine if gene silencing is involved in tristeza cross-protection. The siRNAs found were in the predominant 22 and 21 nucleotide-size classes suggesting an origin from dicer-like (DCL) homologues as was found in virus-silenced Arabidopsis thaliana plants (DCL-2 and DCL-4). Further, viral small interfering RNA showed an association to U known to stabilize Argonaute 1 in the RNA-Induced Silencing Complex. Thus, cross-protection of CTV may be due, in part, to RNA interference.
2. Susceptibility of citrus to leafhopper-borne spread of Spiroplasma citri, causal agent of stubborn disease. It is not known whether rouging stubborn-infected citrus trees in the orchard reduces spread of citrus stubborn disease. ARS researchers at Parlier, CA, used real time Polymerase Chain Reaction assay to test thousands of citrus trees in epidemiology plots for presence of S. citri DNA. A sampling protocol estimating disease incidence was validated and data used to show disease spread patterns were from primary spread with no evidence of secondary spread. This is the first experimental evidence to support the hypothesis that growers need not to rogue stubborn-diseased trees to reduce spread. Rather, focus should be to removing weed hosts of the leafhopper vector and non-citrus reservoir host of the pathogen.
3. Citrus bud graft propagation was evaluated as a means for accidental spread Spiroplasma citri, causal agent of stubborn disease. Certain leafhoppers harbor and transmit S. citri to citrus but it is unknown if natural spread or unintentional spread of stubborn infected trees result in high incidence of disease in the field. ARS researchers at Parlier, CA, evaluated graft transmission of S. citri from seven varieties of Navel orange was very low (0.7%). This low rate cannot explain observed infection rates or pathogen distribution in the field. Therefore, it appears that S. citri spread is mainly via spread by leafhoppers carrying the pathogen that infect citrus during feeding. These results will guide disease management strategies.
Yokomi, R.K., Saponari, M. 2011. Molecular analysis among MCA13-reactive isolates reveals a rapid strategy for assessment of Citrus tristeza virus severity. Acta Horticulturae. 892:251-256.