Citrus variegated chlorosis (CVC), a serious disease affecting sweet orange (Citrus sinensis Osb.) groves in São Paulo and Minas Gerais States, Brazil, was first observed in 1987 and found to be associated with a Gram‐negative, xylem‐limited bacterium (GVG‐B). Electron microscopic studies revealed that the bacterium may appear in situ evenly distributed in the lumen of the tracheary elements and, occasionally, embedded in a lucent matrix adhering to the inner surface of the cell wall elements. GVG‐B measured 1–3.5 μm × 0.3–0.5 μ m and appeared to be at different developmental stages; fibril‐like structures were seen attached to bacteria. GVG‐B rippled wall showed a periodical or annulated structural arrangement with thickness from 25–35 nm in the furrows and 45–55 nm in the ridges. The wall consisted of 3 layers, i.e., an outer, an inner (each comprised of 3‐layered unit membrane structure) and a middle peptidoglycan layer. DNA‐like threads and ribosome‐like granules were present in the cytoplasm. GVG‐B is structurally and morphologically similar to Xylella fastidiosa of which it could be a citrus strain or a strain from other hosts adapted to citrus. Zusammenfassung Elektronenmikroskopische Untersuchungen eines im Xylem‐limitierten Bakteriums bei Süßorangen in Brasilien, die mit citrus variegated chlorosis disease infiziert sind Citrus variegated chlorosis (CVC), eine bedeutende Citruskrankheit bei Süßorangen (Citrus sinensis Osb.) in Plantagen in den brasilianischen Staaten Sao Paulo und Minas Gerais, wurde zum ersten Mai 1987 beobachtet, auch wurde ein Zusanimenhang mit einem Gram‐negativen, im Xylemlimitierten Bakterium (CVC‐B) festgestellt. Elektronenmikroskopische Untersuchungen haben gezeigt, daß das Bakterium in situ gleichmäßig im Lumen der Leitelemente verteilt sein kann, und auch manchmal in einer klaren, an der inneren Oberfläche der Zellwandelemente anhaftenden Matrix eingebettet. CVC‐B maß 1–3,5 μm × 0,3–0,5 μm und schien in unterschiedlichen Entwicklungsstadien zu sein; fibrilähnliche Strukturen waren mit den Bakterien verbunden. CVC‐B hat eine gekräuselte Wand, die eine regelmäßge oder geringelte strukturelle Anordnung zeigte. Die Wandstarke betrug 25–35 nm in den Rillen und 45–55 nm bei den Kämmen. Die Wand bestand aus 3 Schichten, d. h. einer aufieren, einer inneren (jede bestehend aus einer 3‐schichtigen Membranstruktur) und einer mittleren Peptidoglycanschicht. Im Cytoplasma befanden sich DNS‐ãhnliche Fäden und Ribosom‐ähnliche Körnchen. CVC‐B ist von der Struktur und der Morphologie mit Xyelella fastidiosaähnlich und könnte ein Stamm für Zitrusarten sein oder ist ein von einem anderen Wirt adaptierter Stamm von X. fastidiosa.

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.

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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.

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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.

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