K. S. Derrick

Citrus leprosis and its status in Florida and Texas: past and present.

According to published reports from 1906 to 1968, leprosis nearly destroyed the Florida citrus industry prior to 1925. This was supported with photographs showing typical leprosis symptoms on citrus leaves, fruit, and twigs. Support for the past occurrence of citrus leprosis in Florida includes: (1) presence of twig lesions in affected orange blocks in addition to lesions on fruits and leaves and corresponding absence of similar lesions on grapefruit; (2) yield reduction and die-back on infected trees; and (3) spread of the disease between 1906 and 1925. Transmission electron microscopy (TEM) examination of tissue samples from leprosis-like injuries to orange and grapefruit leaves from Florida in 1997, and fruits from grapefruit and sweet orange varieties from Texas in 1999 and 2000 did not contain leprosis-like viral particles or viroplasm inclusions. In contrast, leprosis viroplasm inclusions were readily identified by TEM within green non-senescent tissues surrounding leprosis lesions in two of every three orange leaf samples and half of the fruit samples obtained from Piracicaba, Brazil. Symptoms of leprosis were not seen in any of the 24,555 orange trees examined across Florida during 2001 and 2002. The authors conclude that citrus leprosis no longer exists in Florida nor occurs in Texas citrus based on: (1) lack of leprosis symptoms on leaves, fruit, and twigs of sweet orange citrus varieties surveyed in Florida; (2) failure to find virus particles or viroplasm inclusion bodies in suspect samples from both Florida and Texas examined by TEM; (3) absence of documented reports by others on the presence of characteristic leprosis symptoms in Florida; (4) lack of its documented occurrence in dooryard trees or abandoned or minimal pesticide citrus orchard sites in Florida. In view of the serious threat to citrus in the U.S., every effort must be taken to quarantine the importation of both citrus and woody ornamental plants that serve as hosts for Brevipalpus phoenicis (Geijskes), B. californicus (Banks), and B. obovatus Donnadieu (Acari: Tenuipalpidae) from countries where citrus leprosis occurs.

A novel protein associated with citrus blight has sequence similarities to expansin

A protein associated with citrus blight (CB), a disease of unknown cause, was partially characterized. The 12 kDa protein, designated p12, is diagnostic of CB and is present in leaves and xylem fluid from roots and stems of CB-affected trees. The protein, and up to six other CB-specific proteins, are readily detected by SDS-PAGE of xylem fluid from CB-affected trees. The partial N-terminal amino acid sequence of p12 was found to be unique based on database searches. A cDNA library from CB-affected root cambium was screened with a 60 bp fragment, obtained by PCR amplification of cDNA with degenerate primers designed using the amino acid sequence of p12, and two clones were selected. These clones were sequenced revealing a 674 nucleotide cDNA with a 393 nt ORF which included sequence predicted by the N-terminal amino acid sequence of p12. The amino acid sequence based on the p12 ORF was found to be up to 49% similar and 31% identical to expansins. Bacterial expression of the cloned ORF, which encodes an 11.8 kDa protein plus an N-terminal hydrophobic signal peptide, produced an immunoreactive protein of the expected size. By northern blot analysis, it was determined that p12 transcripts are present in root and stem cambium, but not in leaves of CB-affected trees, suggesting transport of the protein to leaves. Southern hybridization analysis of citrus genomic DNA indicated that p12 is a citrus encoded protein.p>

Citrus blight and other diseases of recalcitrant etiology

Several economically important diseases of unknown or recently determined cause are reviewed. Citrus blight (CB), first described over 100 years ago, was shown in 1984 to be transmitted by root-graft inoculations; the cause remains unknown and is controversial. Based on graft transmission, it is considered to be an infectious agent by some; others suggest that the cause of CB is abiotic. Citrus variegated chlorosis, although probably long present in Argentina, where it was considered to be a variant of CB, was identified as a specific disease and shown to be caused by a strain of Xylella fastidiosa after if reached epidemic levels in Brazil in 1987. Citrus psorosis, described in 1933 as the first virus disease of citrus, is perhaps one of the last to be characterized. In 1988, it was shown to be caused by a very unusual virus. The cause of lettuce big vein appears to be a viruslike agent that is transmitted by a soilborne fungus. Double-stranded RNAs were associated with the disease, suggesting it may be caused by an unidentified RNA virus. Rio Grande gummosis, dry rot root, peach tree short life, and some replant diseases may be diseases of complex etiology. Various microorganisms have been isolated from trees with these diseases, but the diseases may be attributable in part to environmental factors. Determination of the cause of these diseases of complex etiology has proven difficult, in part, because they affect only mature trees.

A Survey for Strains of Xylella fastidiosa in Citrus Affected by Citrus Variegated Chlorosis and Citrus Blight in Brazil

Polymerase chain reaction amplification of DNA from various strains of Xylella fastidiosa with tRNA consensus primers produced three different fingerprint groups. The citrus variegated chlorosis (CVC) and mulberry leaf scorch strains were unique and readily separated from each other and all other strains tested. Internal primers were designed based on the sequence of a DNA fragment unique to the CVC strain. An assay was developed with a mixture of these primers and those reported to detect 18 strains of X. fastidiosa. The assay was used to survey citrus in Brazil. The strain identified to be the cause of CVC was found in constant association with trees with CVC symptoms. On occasion, trees with no symptoms were found to have the CVC strain; this was presumably due to presymptomatic infections. No other strains were found in this survey, and X. fastidiosa was not associated with citrus blight.

Proteins associated with citrus blight.

(...)Complex patterns of proteins from healthy and diseased trees were observed in extracts prepared by pulling buffers through sections of roots under wacuum. Several proteins present in extracts from diseased trees were either absent or present in much lower concentrations in healthy trees. The additional proteins observed from trees with blight appeared to be diagnostic for the disease. Protein patterns characteristic of trees with blight were obserced in assays of 17 blight-symptomatic trees that had been infected by root grafting

Brevipalpus Mites as Vectors of Unassigned Rhabdoviruses in Various Crops

For some time, a limited number of species within the superfamily Eriophyoidea have been the only known acari involved in vectoring viruses to plants (Keifer, 1952; Slykhuis, 1963; Walkey, 1991; Oldfield and Proeseler, 1996). More recently, other acarine families have been incriminated as vectors of plant viruses including Brevipalpus species within the Tenuipalpidae and Petrobia latens (Muller) in the Tetranychidae. The spider mite vectors Barley yellow streak mosaic virus (Robertson, 1987; Robertson and Carroll, 1988; Brunt et al., 1996). This is the only documented instance of a spider mite species involved in the transmission of a viral pathogen. Species within the genus Brevipalpus are considered to be the most important economic pests within the family Tenuipalpidae, especially B. californicus (Banks), B. obovatus Donnadieu and B. phoenicis (Geijskes). All three of these species have been incriminated as vectors of at least one unassigned rhabdovirus. The recent establishment of citrus leprosis virus in Panama and its possible spread to other countries within Central America have increased awareness of the potential threat to citrus in North America and elsewhere in the world. Our limited understanding of Brevipalpus mite vectors, their biologies, feeding damage and virus movement within the mite vectors exacerbate the development of effective control strategies for citrus leprosis. These factors and an increasing list of other unassigned rhabdoviruses that are vectored by Brevipalpus mites has led to the preparation of this special issue to provide a concise update on their status. In the opening paper, Childers, French and Rodrigues provide a review of the biology, feeding injury and economic importance of four Brevipalpus species on selected plants of horticultural importance. Extensive color photographs are provided to clearly illustrate the types of feeding injury associated with these mites. The second paper by Childers, Rodrigues and Welbourn reports on the extensive host plant lists of B. californicus, B. obovatus, and

Introduction of a citrus blight-associated gene into Carrizo citrange [Citrus sinensis (L.) Osbc. × Poncirus trifoliata (L.) Raf.] by Agrobacterium-mediated transformation

The protein p12 accumulates in leaves of trees with citrus blight (CB), a serious decline of unknown cause. The function of p12 is not known, but sequence analysis indicates it may be related to expansins. In studies to determine the function of p12, sense and antisense constructs were used to make transgenic Carrizo citrange using an Agrobacterium-mediated transformation system. Homogeneous β-glucuronidase+ (GUS+) sense and antisense transgenic shoots were regenerated using kanamycin as a selective agent. Twenty-five sense and 45 antisense transgenic shoots were in vivo grafted onto Carrizo citrange for further analyses. In addition, 20 sense and 18 antisense shoots were rooted. The homogeneous GUS+ plants contained either the p12 sense or antisense gene (without the intron associated with the gene in untransformed citrus) as shown by PCR and Southern blotting. Northern blots showed the expected RNA in the sense and antisense plants. A protein of identical size and immunoreactivity was observed in seven of nine sense plants but not in nine antisense or non-transgenic plants. At the current stage of growth, there are no visual phenotypic differences between the transgenic and non-transgenic plants. Selected plants will be budded with sweet orange for field evaluation for resistance or susceptibility to CB and general rootstock performance.

Identification of Strains of Citrus tristeza virus by Subtraction Hybridization

Citrus sudden death (CSD) appears to be a new disease that is a serious problem in Brazil. Symptoms of CSD include yellow stain in the phloem of the rootstock. The cause is not known, but it appears to be infectious and may only affect trees budded on Rangpur lime. In a survey in Brazil, in addition to CSD, we observed numerous trees on Rangpur lime that were obviously declining but had remained in production for several years. Trees with this disease, referred to as Rangpur lime decline (RLD) were different from those with citrus blight (CB). They had near-normal size fruit compared with the small fruit associated with CB and were negative in the serological test for the CB-associated protein (p12). Moreover, they did not have the yellow stain symptom and obviously were declining much more slowly than was reported for CSD. To determine what viruses or virus strains might be associated with CSD, double-stranded (ds)RNAs from fibrous roots of a tree with CSD and stem bark from greenhouse trees infected with Citrus tristeza virus (CTV) isolates T30 and T36 were used to make random primed cDNAs. A Clontech PCR-Select cDNA Subtraction Kit was used to subtract the CSD cDNA with cDNA from an equal mixture of dsRNA from T30 and T36. Of 28 clones that were sequenced, five were found to be significantly different from published CTV sequences. One clone (SDA-1) was found to be only 48% similar to CTV T30 based on amino acid sequence. Using samples collected in October 2001, hybridization assays with a DIG probe of SDA-1 were positive for RNA from roots of declining trees from an area where CSD is reported to occur and from a second area where trees were declining with what had been thought to be CB and are now considered to be RLD. The SDA-1 probe reacted weakly or not at all with RNA from stem bark of trees with CSD, collected in October 2001, or RNA from roots of trees that were declining with CB. Using samples collected in March 2003 from trees with severe decline (nearly dead), the SDA-1 probe reacted with all preparations from both stems and roots. Reactions to the SDA-1 probe also were observed in many stem or root samples from trees with RLD, with early symptoms of CSD, and nonsymptomatic trees. The SDA-1 probe did not react with samples from roots or stems of healthy or CB trees from Florida.

Citrus blight found in Yucatan, Mexico.

Citrus blight, a serious tree decline problem of unknown cause in humid citrus-growing areas such as Florida and Louisiana, South America, South Africa, and the Caribbean, has never been reported from Mexico. Citrus blight has no reliable visual symptoms, and physical and chemical tests have to be used for diagnosis. We used water injection into the trunk (2), zinc and potassium analysis of the outer trunk wood (3), and an immunological test for specific blight proteins in the leaves (1). Low uptake when water is injected into the trunk, and high zinc and potassium in the wood, compared with healthy trees, are characteristic of citrus blight (3). Water injection tests and wood analysis of four healthy and four declining trees in the Dzan, Yucatan, Mexico, area in August 1995, showed highly significant differences in water uptake (healthy trees 44.3 ml/min, declining trees 1.0 ml/min), little difference in wood zinc (healthy 2.8 μg/g, declining 3.0 μg/g) and 39% more potassium in the wood (healthy 0.147%, declining 0.204%). No leaf protein tests were done at this location. Tests on eight declining and five healthy trees in Seye, Yucatan, Mexico, in June 1996, showed significant (P = 0.01 to 0.05) differences in water uptake (healthy 12.9 ml/min, declining 0.6 ml/min), in wood zinc (healthy 2.0 μg/g, declining 7.0 μg/g) and potassium in the wood (healthy 0.156%, declining 0.251%). Leaf samples from all eight declining trees were positive for blight in a specific protein test (1). The visual symptoms of all declining trees tested were the same as those of blight-affected trees in Florida and Cuba: zinc deficiency symptoms in the leaves, thin foliage, wilt, and sprouting from the trunk and the main branches. The reasons for the lack of earlier reports of citrus blight from Mexico are apparently climatic and rootstock related. Many of Mexicos citrus-producing areas are dry and blight does not occur in dry areas such as the Mediterranean countries and California. Most of Mexicos citrus is grown on sour orange (Citrus aurantium L.) rootstock that is highly resistant to citrus blight, but very susceptible to tristeza virus disease. In response to warnings that tristeza disease might appear in Yucatan, growers planted Valencia orange (C. sinensis (L.) Osbeck) on Cleopatra mandarin (C. reticulata Blanco) rootstock in Dzan and on Volkamer lemon (C. limon (L.) N. L. Burm.) in Seye, both highly susceptible to citrus blight (4). Changes in rootstock to avoid one disease led to problems with another. References: (1) K. S. Derrick et al. Proc. Fla. State Hortic. Soc. 105:26, 1993. (2) R. F. Lee et al. Plant Dis. 68:511, 1984. (3) H. K. Wutscher and C. J. Hardesty. J. Am. Soc. Hortic. Sci. 104:9, 1979. (4) R. H. Young et al. Proc. Fla. State Hortic. Soc. 91:56, 1978.

First Report of Citrus Blight in Costa Rica

Citrus blight (CB), causing a chronic decline of citrus, has been an important disease in Florida for over 100 years. CB was first reported in Brazil in the 1980s and is now responsible for the removal of nearly 10% of the trees from production annually. No causal agent has been identified, but CB has been root-graft transmitted to healthy trees, suggesting that the causal agent is infectious (3). Since 1997, CB symptoms were observed in several groves in northern Costa Rica, the most important citrus area of approximately 25,000 ha. Symptoms observed include a general decline and wilt of the tree canopy, off-color leaves, leaf drop, twig dieback, small fruit, delayed blossom, poor growth, and death. A survey near Guanacaste revealed CB symptoms in 7-yr-old Valencia and Pineapple orange trees (Citrus sinensis (L.) Osbeck) grafted on Carrizo citrange (C. sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf.) rootstock. Since 1997, 6% of the trees in this area have been replanted annually because of CB symptoms. Similar situations were observed in other groves in the northern citrus area. Dot immunobinding assays (DIBA) (1) were used to detect the P12 protein associated with CB with 20 of 22 trees showing CB-like symptoms giving a positive test. Zinc (Zn) accumulation in trunk wood and water uptake tests were done according to Roistacher (2) in 8 healthy and 20 symptomatic trees which were positive for CB using DIBA. The average Zn concentration of 16 declining trees was 4.6 ± 1.9, whereas the average concentration for 8 healthy trees was 2.0 ± 0.9. The average water uptake in 1 min was 14 ml for healthy trees, and virtually zero for the 20 symptomatic trees. These diagnostic tests confirm the presence of CB in the northern citrus area of Costa Rica, and the surveys indicate the disease is beginning to spread and become economically important. To our knowledge, this is the first report of CB in commercial citrus in Costa Rica. References: (1) K. S. Derrick et al. Plant Dis. 74:168, 1990. (2) C. N. Roistacher. Pages 57-66 in: Graft-Transmissible Diseases of Citrus. Handbook for Detection and Diagnosis. C.N. Roistacher, ed. Food and Agriculture Organization, Rome, 1991. (3) D. P. H. Tucker et al. Plant Dis. 68:979, 1984.