During the 2017/18 growing season, a new and damaging disease of squash was observed in several commercial plantations in the Northern Cape Province of South Africa. Soft, water-soaked lesions, 3 to 6cm in diameter, were present on the rind of fruits . Many fruits were rotting inside . A total of 50 to 80% of fruits were affected per field.
Soft rot of numerous crops is usually caused by Pectobacterium and Dickeya species of bacteria. However, from all diseased fruits, an almost pure culture of fluorescent bacteria was isolated on Milk-Tween and King’s B media. Isolates were identified as Pseudomonas syringae based on the LOPAT tests. Several pathovars of Pseudomonas syringae have been reported to cause foliar blights and fruit spots. Usually, the outbreaks of foliar diseases on cucurbits are assumed to be caused by P. syringaep v. lachrymans.
Squash fruits showing the rot symptoms were collected from four fields in the Northern Cape. Isolations were done from the soft, water soaked lesions. Tissue from lesion margins of infected fruits was removed with a sterile scalpel and macerated in 1ml of sterile distilled water for 15 minutes at 25oC. Plant extracts were streaked onto NA, CVP and Milk-Tween media.
Bacterial growth on plates was evaluated after four days of incubation at 25oC. Suspected colonies were purified by streaking onto King’s B. Production of fluorescent pigment on King’s B agar medium, Gram stain reaction, production of levan, oxidase activity, ability to cause potato rot, arginine utilisation and production of a hypersensitive reaction on tobacco (LOPAT tests) were determined for each bacterial strain.
Gram negative bacteria fluorescent under UV light on MilkTween medium were consistently isolated from diseased tissues. They were Levan-positive, oxidase, potato rot, and arginine dihydrolase negative. All induced HR reaction in tobacco leaves and were tentatively identified as P. syringae.
Genomic DNA of four strains isolated from squash fruits was extracted with the Wizard Genomic DNA purification Kit (Promega, Madison, USA) according to the manufacturer’s instructions. The primers used for PCR amplification and sequencing of the cts and gyrB genes were developed by Hwang et al. (2005, App Environ Microbiol 71:5182-81). PCR amplification was done as reported by Morris et al. (2008, Int Soc MicrobiolEcol 2:321-34).
Sequencing was performed by Inqaba Biotechnology (Pretoria, South Africa). The cts and gyrB nucleotide sequences obtained in this study were aligned with sequences of eleven Pseudomonas syringae/savastanoi reference strains from the Plant Associated and Environmental Microbes Database using the MAFFT 7 online alignment tool. Phylogenetic trees were constructed with the neighbour-joining method and evolutionary distances calculated according to the method of Kimura. Bootstrap analysis of the data, based on 1 000 permutations was used to assess the stability of relationships. The trees were viewed and edited using MEGA 7.
A BLAST search of the EMBL/GenBank database conducted with the sequences revealed a high degree of sequence identity (97 – 99%) with previously determined sequences of P. syringaep v.syringae. Figure 2 shows the phylogenetic relationship derived from a neighbour-joining analysis of the pairwise comparison among the gyrB and cts sequences of four strains from this study with ten sequences of described pathovars of P. syringae. Pseudomonas chicorii was used as an outgroup taxon. Phylogenetic trees constructed for two genes were identical. Four strains isolated from rotting squash fruits grouped with P. syringaep v. syringae.
Pathogenicity screening of four strains was performed on the store-bought squash fruits. A sterile needle was dipped into a bacterial colony of the strain growing on King’s B and then inserted under the epidermis of a fruit. Three fruits were used per strain. Pathogenicity tests were repeated. Three fruits were used for the negative control treatment (sterile water). Inoculated fruits were placed in a sterile plastic box, incubated at 25°C and observed daily for the development of symptoms. Bacteria were re-isolated from the developing lesions on King’s B and confirmed by colony morphology on King’s B, fluorescence under UV light and LOPAT tests.
Four strains induced water soaked spots when inoculated directly into squash fruits three days after inoculation. As the disease progressed, the spots enlarged, became soft and rot spread inside the fruits two weeks after inoculation. Fruits inoculated with sterile water did not develop any symptoms. From all symptomatic fruits, fluorescent P. syringae was isolated and confirmed by LOPAT, thus fulfilling Koch’s postulates.
A new disease on squash in South Africa is reported, soft rot of fruits, caused by Pseudomonas syringaepv. syringae. Koch’s postulates were confirmed using four strains, which were subjected to further taxonomic investigations. Morphological and biochemical characterisation using the Gram-stain, colony morphology, fluorescence and LOPAT tests showed that the four strains from squash had similar biochemical characteristics and resembled those of P. syringae. The gyrB and cts sequences of all isolates were 98 to 99% homologous to that of P. syringaepv. syringae and all clustered with P. syringaepv. syringae in neighbour-joining phylogenetic trees. We conclude that the bacteria associated with the soft rot of squash fruits in South Africa are P. syringaepv. syringae. – Press release
