Development of a lateral flow device for in‐field detection and evaluation of PCR‐based diagnostic methods for Xanthomonas campestris pv. musacearum,the causal agent of banana xanthomonas wilt

Xanthomonas campestris pv. musacearum (Xcm) is the causal agent of banana xanthomonas wilt, a major threat to banana production in eastern and central Africa. The pathogen is present in very high levels within infected plants and can be transmitted by a broad range of mechanisms; therefore early specific detection is vital for effective disease management. In this study, a polyclonal antibody (pAb) was developed and deployed in a lateral flow device (LFD) format to allow rapid in‐field detection of Xcm. Published Xcm PCR assays were also independently assessed: only two assays gave specific amplification of Xcm, whilst others cross‐reacted with non‐target Xanthomonas species. Pure cultures of Xcm were used to immunize a rabbit, the IgG antibodies purified from the serum and the resulting polyclonal antibodies tested using ELISA and LFD. Testing against a wide range of bacterial species showed the pAb detected all strains of Xcm, representing isolates from seven countries and the known genetic diversity of Xcm. The pAb also detected the closely related Xanthomonas axonopodis pv. vasculorum (Xav), primarily a sugarcane pathogen. Detection was successful in both naturally and experimentally infected banana plants, and the LFD limit of detection was 10⁵ cells mL^?1. Whilst the pAb is not fully specific for Xcm, Xav has never been found in banana. Therefore the LFD can be used as a first‐line screening tool to detect Xcm in the field. Testing by LFD requires no equipment, can be performed by non‐scientists and is cost‐effective. Therefore this LFD provides a vital tool to aid in the management and control of Xcm.     

Survival of Xanthomonas campestris pv. campestris in the phyllosphere and rhizosphere of weeds

The phyllosphere and rhizosphere of weeds are important niches for phytobacterial survival. The absence of information in Brazil regarding Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot in crucifers, motivated this study. Twenty‐six weed species belonging to 14 botanical families were included in field experiments between August 2014 and October 2015. Lepidium virginicum and Raphanus raphanistrum (Brassicaceae) demonstrated great potential for survival of Xcc in the phyllosphere, with the bacterium isolated after 56 and 70 days, respectively. Low variation between maximum and minimum temperatures, high rainfall and high relative humidity at specific times of the year contributed to longer Xcc survival periods in the phyllosphere of some species. Xcc survived in the rhizosphere only in R. raphanistrum, where it was isolated for up to 28 days. No relation was found between climatic factors and survival in the rhizosphere. The data indicate that control of brassicaceous weeds will contribute to the control of black rot.     

Multiplex PCR for specific and robust detection of Xanthomonas campestris pv. musacearum in pure culture and infected plant material

The present study developed a pathovar‐specific PCR for the detection of Xanthomonas campestris pv. musacearum (Xcm), the cause of banana xanthomonas wilt, by amplification of a 265‐bp region of the gene encoding the general secretion pathway protein D (GspD). A distinct DNA fragment of the expected size was amplified from genomic DNA from all of 12 Xcm isolates tested and no amplification of DNA was observed from other xanthomonads or plant‐associated bacteria, including the two closely related species Xanthomonas vasicola pv. holcicola and Xanthomonas axonopodis pv. vasculorum. The Xcm‐specific PCR was successfully multiplexed with internal control primers targeting 16S rDNA for application on DNA from bacterial cultures and with primers targeting plant mitochondrial 26S rDNA for application on DNA extracted from plant material. Diagnostic discrimination of healthy and infected plants was subsequently demonstrated in tests on artificially inoculated screenhouse cultivars of banana and field bananas with and without symptoms sampled from different parts of Uganda. This study therefore demonstrated a robust and specific Xcm diagnostic tool with the added advantage of applying internal PCR controls for direct quality assessment of results.     

Application of polyclonal and monoclonal antibodies for the detection of Xanthomonas campestris pv. campestris in crucifer seeds using immunofluorescence microscopy

Polyclonal and monoclonal antibodies (PCAs and MCAs) were tested for the detection ofXanthomonas campestris pv.campestris (Xcc) in cabbage seeds using immunofluorescence microscopy (IF). It was concluded that PCA 94, MCAs 20H6, 2F4, 18G12 and a mixture of MCAs 20H6, 18G12, 2F4 and 16B5 could be used to detect Xcc in seed extracts when 5 min and 2.5 h shaking of seeds are used as extraction methods. The reliability of confirming suspect colonies with MCAs and PCA 94 in IF depended in part on the seed lot tested and the antibody used. Some virulent Xcc strains derived from seed lots, did not react with MCAs 10C5, 2F4, 18G12, 17C12 and 16B5. On the other hand, saprophytic isolates obtained from one seed lot cross-reacted with MCA 17C12 and to a lesser extent with MCAs 2F4, 18G12 and PCA 94. No relationship was found between IF-reactions of Xcc strains using MCAs and reactions of Xcc strains in pathogenicity testing. Xcc andX. c. pv.amoraciae (Xca) could in general not be distinguished on the basis of reactions with MCAs and PCAs. Also in pathogenicity tests Xcc and Xca were hard to distinguish.     

Genome‐wide identification of pathogenicity genes in Xanthomonas oryzae pv. oryzae by transposon mutagenesis

A transposon mutant library was constructed from the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) KACC10331 by Tn5 transposon mutagenesis. The susceptible rice cultivar Milyang 23 was inoculated with a total of 24 540 mutants resistant to kanamycin and 67 avirulent or reduced‐pathogenicity mutant strains were selected for study. Southern hybridization verified that 84 mutant strains had single‐copy insertions and their single‐transposon insertion sites were identified by sequencing analysis combined with thermal asymmetric interlaced (TAIL)‐PCR. The single‐transposon‐tagged sequences of 21 mutant strains belonged to pathogenicity‐related genes previously reported in Xanthomonas species, while the other 46 single‐transposon‐tagged sequences included diverse functional genes encoding, five cell‐wall‐degrading enzymes, three fimbrial and flagella assembly regulators, five regulatory proteins, 15 metabolic regulators and 18 hypothetical proteins, which were identified as novel pathogenicity genes of Xoo.     

Comparison of immunofluorescence microscopy and dilution-plating for the detection of Xanthomonas campestris pv. campestris in crucifer seeds

The correlation between immunofluorescence microscopy (IF) and dilution-plating on nutrient starch cycloheximide agar (NSCA) or NSCA with the addition of nitrofurantoin and vancomycin (NSCAA) was studied for the detection ofXanthomonas campestris pv.campestris (Xcc) in crucifer seeds. When checking 50 l of the seed extract in IF, IF and dilution-plating gave corresponding results (both positive or negative) for 45.4–56.4% of the samples tested. No differences were observed in this respect between tests using a polyclonal antiserum (PCA 94) and replicate tests using monoclonal antibodies (MCA 20H6). When 20 l of the seed extract was checked in IF, 67.3–71.3% of the samples tested were both positive or negative with dilutionplating and IF. IF negative and dilution-plating positive samples were found for 0.0–7.3% of all samples tested. The percentage of IF positive and dilution-plating negative samples ranged from 26.7–29.2 (20 l seed extract checked) to 41.8–47.3% (50 l seed extract checked). Generally, the probability of isolating Xcc increased with increasing numbers of fluorescent cells found in IF. Above 10 000 cells per ml the probability of isolating Xcc ranged from 57.1–81.8%. Increasing the extraction time from 5 min to 2.5 h shaking showed no significant increase of the number of samples found positive in IF and dilution-plating. However, when using both 5 min and 2.5 h shaking as compared to 5 min shaking only, more samples can be found positive in IF (1.0–14.5%) and dilution-plating (3.0–18.5%). Examining 1 l instead of 50 l of the sample smear, would increase the correspondence between IF and dilution-plating results up to minimally 69.1% (MCA 20H6). However, the risk of false-negative results in IF as compared to dilution-plating would also increase.     

Effect of burning and high temperature on survival of Xanthomonas translucens pv. pistaciae in infected pistachio branches and twigs

This paper reports the efficacy of burning and heat‐treating pistachio branches and twigs as a means of disposing of prunings from trees infected with Xanthomonas translucens pv. pistaciae (Xtp). Burning of pistachio wood, naturally infected with Xtp, was conducted twice under field conditions. Viable Xtp was detected in some non‐burned wood, but not in charcoal, ash or partially burned wood. Controlled laboratory experiments were conducted with pure cultures of Xtp and naturally and artificially infected pistachio wood. In liquid culture, 65°C was lethal to Xtp, whereas survival at 60°C or less varied with culture medium and duration of exposure. Xtp survived in infected wood exposed to 40–55°C for at least 60 min but was killed by exposure to 60°C for 15 min or more. Overall, the results of burning and heat treatment were consistent, and confirmed that burning was a reliable eradication technique to dispose of infected wood, such as prunings, providing the pathogen was exposed to a temperature of 60°C or greater for at least 15 min.     

Movement of Xanthomonas campestris pv. vitians in the stems of lettuce and seed contamination

Xanthomonas campestris pv. vitians , the causal agent of bacterial leaf spot of lettuce (BLS), can be seedborne, but the mechanism by which the bacteria contaminates and/or infects lettuce seed is not known. In this study, the capacity of X. campestris pv. vitians to enter and translocate within the vascular system of lettuce plants was examined. The stems of 8- to 11-week-old lettuce plants were stab-inoculated, and movement of X. campestris pv. vitians was monitored at various intervals. At 4, 8, 12 and 16 h post-inoculation (hpi), X. campestris pv. vitians was recovered from 2 to 10 cm above (depending on stem length) and 2 cm below the inoculation site. Xanthomonas campestris pv. vitians was also recovered from surface-disinfested stem sections of spray-inoculated plants. Together, these results are consistent with X. campestris pv. vitians invading and moving systemically within the vascular system of lettuce plants. To investigate the mechanism of seed contamination, lettuce plants at the vegetative stage of growth were spray-inoculated with X. campestris pv. vitians and allowed to develop BLS. Seed collected from these plants had a 2% incidence of X. campestris pv. vitians external colonization, but no bacteria were recovered from within the seed.     

Xanthomonas campestris pv. populi,the causal agent of bark necrosis in poplar

A bacterium was isolated from superficial bark necroses on young poplars and its pathogenicity demonstrated by inoculation experiments. The organism was identified asXanthomonas campestris. Cross-inoculations showed that a previously undescribed pathovar was involved. It is suggested to designate this organismX. campestris pv.populi.Samenvatting Uit een oppervlakkige bastnecrose bij jonge populieren werd massaal een bepaalde bacterie geïsoleerd. Met deze bacterie werden gezonde populieren in het veld geïnoculeerd via verwonding van de bast. Als gevolg van de inoculaties ontwikkelden zich bij ongeveer 40% van de geïnoculeerde bomen hetzelfde type bastnecrosen, terwijl bij de controleplanten geen enkele reactie optrad. Uit de kunstmatig verkregen necrosen werd dezelfde bacterie geïsoleerd.Identificatie met biochemische en serologische methoden toonde aan dat de bacterieXanthomonas campestris was.Vervolgens werden in de kas kruisinoculaties uitgevoerd met verschillende xanthomonaden op populier, wilg, kool en geranium. DeX. campestris isolaten uit populier tastten behalve populier ook wilg aan. De andere gebruikte stammen waren waardplant-specifiek, al bleven sommigen ervan minstens acht maanden in leven in een niet-waardplant, evenwel zonder symptomen te veroorzaken. Geconcludeerd wordt, dat de bastnecrosen zijn veroorzaakt door een nog niet beschreven pathovar vanX. campestris. Voorgesteld wordt om deze bacterieXanthomonas campestris pv.populi te noemen.