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Title: Serratia marcescens, a phloem-colonizing, squash bug-transmitted bacterium: Causal agent of cucurbit yellow vine disease

Author
item Bruton, Benny
item Mitchell, F.
item Fletcher, J.
item Pair, Sammy
item Wayadande, A.
item Melcher, U.
item Brady, J.
item Bextine, B.
item Popham, Thomas

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/26/2003
Publication Date: 8/1/2003
Citation: Bruton, B.D., Mitchell, F., Fletcher, J., Pair, S.D., Wayadande, A., Melcher, U., Brady, J., Bextine, B., Popham, T.W. Serratia marcescens, a phloem-colonizing, squash bug-transmitted bacterium: Causal agent of cucurbit yellow vine disease. Plant Disease. 2003. 87:937-944.

Interpretive Summary: In 1988, Cucurbit yellow vine disease (CYVD) was first observed in squash and pumpkin in Central Texas and Oklahoma. In 1991, the disease was detected in watermelon and melon. Since then, the disease has also caused substantial damage to cucurbits in Arkansas, Tennessee, and Massachusetts. The diagnostic characteristic of CYVD affected plants is the honey-brown phloem discoloration in the crown and primary root. The objectives of the study were to isolate the phloem-inhabiting bacterium, develop an effective inoculation proceedure, prove Koch's Postulates, and determine the ability of squash bugs to transmit the bacterium to plants. Using PCR with CYVD-specific primers to confirm identity of the the CYVD pathogen, the bacterium was isolated on nutrient agar. Koch's postulates were completed for cucurbit yellow vine disease, demonstrating that a strain of the cosmopolitan bacterium, Serratia marcescens, is the causal agent. Although S. marcescens is readily cultivated from various ecological niches, its cultivation from CYVD-affected cucurbits is significant because it represents the first successful growth of a phloem-colonizing bacterium on common laboratory media. The development of an effective mechanical inoculation method for S. marcescens on cucurbits was an essential step in this work. Infection rate was highly dependent on plant growth stage when inoculated. To achieve maximum disease development, plants should be inoculated when the seedlings first emerge and prior to cotyledons unfolding. Evidence presented from our field studies confirmed that the squash bug can transmit S. marcescens, the CYVD causal bacterium. The S. marcescens-Anasa tristis relationship described here is, to our knowledge, the first instance in which the squash bug has been identified as a vector of a plant pathogen.

Technical Abstract: Cucurbit yellow vine disease (CYVD), which can inflict heavy losses to watermelon, pumpkin, cantaloupe and squash in U. S. production areas from the mid-west to northeast, causes phloem discoloration, foliar yellowing, wilting, and plant decline. By electron microscopy, the disease had been previously associated with the presence in phloem sieve tubes of a walled bacterium. Bacteria were cultured from the phloem of crown sections of symptomatic plants of all four cucurbit species. Those bacteria testing positive in CYVD-specific PCR were all Gram negative and appeared morphologically identical, producing creamy white, smooth, entire, convex colonies on Luria-Bertani (LB) or nutrient agar. The bacteria were identified, by nucleotide sequence analysis of the 16S rDNA genes, to be Serratia marcescens. Characterized cucurbit-derived isolates of S. marcescens were introduced into greenhouse-grown squash plants by puncture inoculation and into field-grown squash plants by enclosure with S. marcescens-fed squash bugs, Anasa tristis. Up to 60% of the bacteria-inoculated plants in the greenhouse and up to 29% of field plants caged with inoculative squash bugs developed phloem discoloration and tested positive for S. marcescens by CYVD-specific PCR. None of the controls developed phloem discoloration or tested positive by PCR. Some of the diseased field plants also yellowed, wilted and collapsed, although, these latter symptoms were not observed in the greenhouse-grown, puncture-inoculated plants. The morphology and pigmentation of bacteria cultured from crown tissue of a subset of plants in each experimental group were indistinguishable from those of the inoculum bacteria. Nucleotide sequence analysis of the PCR products amplified from several of the samples in each group, as well as of 16S rDNA genes of bacteria cultured from the symptomatic test plants, demonstrated that these re-isolated bacteria were identical to those of the S. marcescens isolates used for the inoculations. Evidence presented from our studies confirms that the squash bug can transmit S. marcescens, the CYVD causal bacterium. The S. marcescens-Anasa tristis relationship described here is the first instance in which the squash bug has been identified as a vector of a plant pathogen. Our experiments represent a completion of the steps of Koch's postulates, demonstrating that S. marcescens is the causal agent of CYVD and that the squash bug, A. tristis, is a vector of the pathogen.