|Yokomi, Raymond - Ray|
Submitted to: International Organization of Citrus Virologists Proceedings
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/29/2008
Publication Date: 5/14/2011
Citation: Mello, A.F., Yokomi, R.K., Fletcher, J. 2011. Assessment of Stubborn Disease Incidence in Citrus. In: Proceedings of the International Organization of Citrus Virologists. p. 123-130. Interpretive Summary: Citrus stubborn disease (CSD) has been a problem in California for over 90 years, yet, methods for rapidly detecting its causal agent, Spiroplasma citri, to assess disease incidence have not been well developed. CSD incidence was assessed in two groves of Navel oranges. Pathogen extraction from fruit columella was more efficient than from leaves, midribs, petioles, or bark for pathogen cultivation. Three sampling procedures were tested for estimating disease incidence: stat sampling (STAT), in which the fifth tree from every fifth row were sampled; hierarchical sampling (HS), in which four trees were sampled as a single unit from 25% of all trees in the field; and each tree (Singles) within 6 blocks of 64 trees per field were sampled. STAT sampling resulted in estimated CSD incidences of 45.9% and 1.3% in groves 1 and 2, respectively, by cultivation. In comparison, HS yielded incidences of 71.4% and 3.6%; whereas Singles resulted in 50% and 1.6% in the two groves. More infected trees were found when the same samples were assayed by PCR than by cultivation. In summary, CSD incidence in grove 1 was confirmed to be high, and low in grove 2. PCR was superior to culturing for detecting CSD and, in the fields sampled, STAT and singles sampling were the most accurate in estimating CSD incidence. As a result, spread of CSD in field trees by S. citri is being monitored using PCR from DNA extracts of fruit columella from individual trees.
Technical Abstract: Citrus stubborn disease (CSD) has been a problem in California for over 90 years, yet, methods for rapidly detecting its causal agent, Spiroplasma citri, for use in estimating disease incidence have not been optimized. Two 8 ha blocks within two commercial groves were sampled in July and August, 2006. Different tissues of sweet orange were tested for spiroplasma cultivation and three sampling procedures for estimating disease incidence were compared using cultivation and polymerase chain reaction (PCR). Receptacle and columella yielded cultivable spiroplasmas more consistently than did leaves, midribs, petioles, or bark. Stat sampling, in which the fifth tree from every fifth row was sampled, resulted in estimated incidences of 45.9 and 1.3% by cultivation of the fruit receptacles in groves 1 and 2, respectively. Hierarchical sampling, in which four trees were sampled as a single unit from 25% of the trees in field, yielded incidences of 71.4 and 3.6% in groves 1 and 2 respectively, by culturing, and 73.3 and 3.6% by PCR. In the third method, all trees within 6 blocks of 64 trees in each grove, sampled individually, yielded incidences of 50 and 1.6% (groves 1 and 2 respectively) by culturing, and 58.4 and 2.1% (groves 1 and 2 respectively) by PCR. Thus, stubborn incidence in grove 1 was confirmed as high and that of grove 2 low. In these tests, PCR was superior to culturing; it is relatively inexpensive, sensitive, and rapid, permitting analysis of a large number of samples.