RESUMEN

Strains of the plant-pathogenic bacterium Xanthomonas axonopodis pv. citri are differentiated into two groups with respect to aggressiveness (normal and weak) on Citrus grandis cultivars but not on other Citrus species such as Citrus sinensis. Random mutagenesis using the transposon Tn5 in X. axonopodis pv. citri strain KC21, which showed weak aggressiveness on a C. grandis cultivar, was used to isolate mutant KC21T46, which regained a normal level of aggressiveness on the cultivar. The gene inactivated by the transposon, hssB3.0, was shown to be responsible for the suppression of virulence on C. grandis. Sequence analysis revealed it to be a new member of the pthA homologs, which was almost identical in sequence to the other homologs except for the number of tandem repeats in the central region of the gene. hssB3.0 appears to be a chimera of other pthA homologs, pB3.1 and pB3.7, and could have been generated by recombination between these two genes. Importantly, in X. axonopodis pv. citri, hssB3.0 was found in all of the tested isolates belonging to the weakly aggressive group but not in the isolates of the normally aggressive group. Isolation of the virulence-deficient mutant KC21T14 from KC21, in which the pathogenicity gene pthA-KC21 was disrupted, showed that hssB3.0 induces a defense response on the host but partially interrupts canker development elicited by the pathogenicity gene in this bacterium.

Asunto(s)

Proteínas Bacterianas/genética , Citrus/microbiología , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Xanthomonas axonopodis/genética , Secuencia de Aminoácidos , Citrus/genética , Genes de Plantas/genética , Inmunidad Innata/genética , Datos de Secuencia Molecular , Familia de Multigenes , Enfermedades de las Plantas/microbiología , Hojas de la Planta/microbiología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Efectores Tipo Activadores de la Transcripción , Virulencia , Xanthomonas axonopodis/patogenicidad

RESUMEN

We developed a multiplex reverse transcription polymerase chain reaction (RT-PCR) to detect six citrus viroids: Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid III (CVd-III), Citrus viroid IV (CVd-IV) and Citrus viroid OS (CVd-OS) and Apple stem grooving virus (ASGV, synonym: Citrus tatter leaf virus (CTLV)) from citrus plants. The multiplex RT-PCR was also designed to distinguish CVd-I-LSS (a distinct variant of CBLVd) from CBLVd. By the multiplex RT-PCR, one to eight fragments specific to the pathogens were simultaneously amplified from one sample and identified by their specific molecular sizes in 6% polyacrylamide gel electrophoresis. The results of the multiplex RT-PCR were consistent with those of other diagnoses, such as uniplex RT-PCR, to detect each of the pathogens. The multiplex RT-PCR provides a simple and rapid method for detecting various viroids and ASGV in citrus plants, which will help diagnose many citrus plants at a time.

Asunto(s)

Citrus/virología , Virus de Plantas/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Electroforesis en Gel de Poliacrilamida , Virus ARN/aislamiento & purificación , Viroides/aislamiento & purificación

RESUMEN

ABSTRACT Sequential polyacrylamide gel electrophoresis analyses showed many viroid-like RNAs in samples collected from citrus trees in Japan. Reverse transcription polymerase chain reaction and sequencing analyses of the amplified fragments verified that they were derived from variants of six citrus viroids, Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd) including CVd-I-LSS (a distinct variant of CBLVd), Hop stunt viroid, Citrus viroid III, Citrus viroid IV, and Citrus viroid OS. The samples induced symptoms with variable severity in Arizona 861-S1 'Etrog' citrons (Citrus medica L.) likely due to the varying accumulation patterns produced by the different viroids. Some of the symptoms caused by the samples harboring the citrus viroids other than CEVd were as severe as those caused by CEVd. Some source citrus trees showing the severe bark scaling characteristic of exocortis disease in trifoliate orange (Poncirus trifoliata (L.) Raf.) rootstocks contained only citrus viroids other than CEVd in complex. This indicates that certain exocortis-like diseases in Japan were caused by some combination of citrus viroids not including CEVd.