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Center Rot of Onion​

​​​​​M​ehra, L. K., Adhikari, U., Ojiambo, P. S., Cowger, C. 2019. Center Rot of Onion. The Plant Health Instructor. DOI: DOI:10.1094/PHI-I-2019-0603-01

DISEASE:​​ Center Rot of Onion

PATHOGEN: Pantoea ananatis, Panto​ea agglomerans, Pantoea alli and Pantoea stewartii subsp. indologene​s​

HOSTS: Onion (Allium cepa L.), garlic (Allium​ sativum L.), shallots (Allium cepa var. aggregatum L.), leeks (Allium ampeloprasum L.), chives (Allium schoenoprasum L.).​

Center rot of onion has great po​tential to cause significant economic losses to onion production, and in severe cases yield losses up to 90% can occur.​

Authors

Gaurav Agarwal1, Spencer Stumpf1, Brian Kvitko2, and Bhabesh Dutta1

1Department of Plant Pathology, Coastal Plain Experiment Station, University of Georgia, Tifton, GA 31793​
2Department of Plant Pathology, University of Georgia, Athens, GA 30602
​Corresponding author: Bhabesh Dutta: bhabesh@uga.edu​​

Symptoms and signs

​​​​​​​​​​​​
​Fig. 1. S​​ymptoms of center rot in onion foliage and bulb; A: White, bleached out, necrotic lesions on onion foliage; B: Necrotic lesions and bulb rot.
Disease symptoms on foliage and onion bulbs caused by four Pantoea species are similar (Gitaitis et al. 2002; Walcott et al. 2002; Edens et al. 2006; Stumpf et al. 2018). Foliar symptoms produced by Pantoe​a sp. complsex start with water-soaked lesions spanning the length of the leaf blade, which gradually become blighted resulting in desiccation and collapse of the tissue. As the disease pr​ogresses in the plant, severe wilting and blighting of foliage can occur leading to complete foliage death (Gitaitis and Gay 1997). Bacterial movement from foliar tissue into bulb was demonstrated experimentally, where it was shown that P. ananatis blade infection could lead to bulb decay (Carr et al. 2013). Additionally, the authors observed that the specific bulb scale showing symptoms could be traced back to the corresponding infected blade (Fig. 1). Therefore, the authors concluded that protecting foliage of onion leaves can protect against bulb incidence of center rot. ​

Host Range and Geographic Distribution

Pantoea ananatis can cause disease on a broad range of plants, including​ many economically important crops, around the globe (Coutinho and Venter 2009). The first record of P. ananatis​ causing plant disease was in 1928 where it was identified causing fruitlet rot on pineapple in Philippines (Serrano 1928). The “ananatis" species name derives from the first host of isolation, pineapple (Ananas comosus).  P. ananatis can survive as an asymptomatic epiphyte or symptomatic endophyte on both dicots and monocots (Coutinho and Venter 2009). Gitaitis et al. (2002) reported that P. ananatis is widely distributed throughout Georgia on numerous weed species commonly found near onion production sites. P. ananatis was first reported in the United States in Georgia in 1997. Since then, it has been identified in other onion growing regions of the United States including Colorado (Schwartz and Otto 2000), Michigan (Schwartz and Mohan 2008), New York (Carr et al. 2010), and Pennsylvania (Pfeufer et al. 2008). Additionally, P. ananatis has been isolated from many economically important crops around the world including honeydew melon in Ecuador (Wells et al. 1987), cantaloupe (Bruton et al. 1991), onion (Gitaitis and Gay 1997), and sudangrass (Azad et al. 2000) in the United States, eucalyptus in South Africa (Coutinho et al. 2002), rice in Italy (Cortesi and Pizzatti 2007) and Russia (Egorova et al. 2015), netted melon in Japan (Kido et al. 2008), maize in Argentina (Alippi and Lopez 2010) and Poland (Krawczyk et al. 2010), and sorghum in Brazil (Cota et al. 2010).

P. agglomerans was first described as a causal agent for the center rot of onion in South Africa (1981). Later, the bacterium was identified as a causal agent of center rot in Georgia in 2006 (Hattingh and Walters. 1981; Edens et al. 2006). This was the first report of the bacterium causing this disease in the United States (Edens et al. 2006).  P. allii was identified as the third member in the Pantoea species complex causing center rot of onion in Georgia (Brady et al., 2011). Recently, P. stewartii subsp. indologenes was included as the fourth member in the center rot complex of onion along with P. ananatis, P. agglomerans and P. allii (Stumpf et al., 2018).​

Pathogen biology and molecular identification

Pantoea ananatis is a Gram-negative, rod-shaped, facultative anaerobe similar to other members of the Enterobacteriales (Kim et al. 2012; Gitaitis and Gay 1997). Colonies are yellow pigmented and the cells utilize glucose in an oxidative and fermentative manner. The bacterium tests positive for the following biochemical assays: β-D-galactosidase and catalase, utilization of citrate, and production of acetoin, and indole.  P. ananatis tests negative for ornithine decarboxylase, lysine decarboxylase, urease, and oxidase (Gitaitis and Gay 1997). Strains of other closely related Pantoea spp., namely P. agglomerans, P. alli, and P. stewartii subsp. indologenes have been isolated that can cause onion center rot symptoms. Identifying the causal agent of center rot infection is important in implementing an effective management strategy, as the epidemiology among the four Pantoea species may be different. In general, P. ananatis and P. alli can be differentiated from P. agglomerans as they produce a positive reaction for indole production and a negative reaction for phenylalanine deaminase and nitrate reductase enzymes. P. ananatis can be differentiated from P. allii based on acid production from amygdalin. P. allii produces acid from amygdalin, while P. ananatis does not (Brady et al. 2011). However, P. stewartii subsp. indologenes cannot be differentiated biochemically from P. ananatis nor P. alli.

The sequencing of 16S rRNA is considered to be the standard method for elucidating phylogenetic relationships among bacterial pathogens. In Pantoea spp., the analysis of the 16S rRNA gene revealed a close phylogenetic relationship among P. agglomerans, P. allii, and P. ananatis, as a result this test is unreliable to distinguish these species. (Hauben et al. 1998; Naum et al. 2008). Gevers et al. (2005) also contradicted the dependability of the 16S rRNA gene as a reliable phylogenetic marker due to the occurrence of potential recombination or horizontal gene transfer events in bacteria that may distort the actual relationship. A more reliable sequence-based method for genotyping strains is multilocus sequence analysis (MLSA). This approach uses the sequences of five to seven housekeeping genes to define sequence profiles of similar strains. Briefly, the 16S rRNA gene sequence may reliably identify strains to the genus level, but not to the correct species level. Sequencing housekeeping genes is the preferred method to differentiate closely related Pantoea species (Gevers et al. 2005).

Accurate identification is fundamental to understand pathogen spread, epidemiology, and management of bacterial pathogens. The current methods of serological techniques and 16S rRNA sequencing may fail to recognize the introduction of exotic strains or the development and spread of strains with enhanced virulence (Barak and Gilberston 2003). Delétoile et al. (2009) characterized 36 Pantoea strains, including 28 strains from diverse origins (hosts, geographical locations, and ecological niches) initially identified as P. agglomerans using MLSA based on six protein-coding genes (fusA, gyrB, leuS, pyrG, rplB, and rpoB), and using biochemical and antimicrobial susceptibility assays. Phylogenetic analysis and subsequent comparison with other Enterobacteriales species revealed that the genus Pantoea is highly diverse. All 20 P. agglomerans strains could be distinguished by multilocus sequence typing, indicating a high power of discrimination by MLSA for strain typing and population structure in this species (Delétoile et al. 2009). The authors also found that biochemical characteristics such as fosfomycin resistance and utilization of d-tartrate could differentiate P. agglomerans from other Pantoea species. Furthermore, repA gene was detected, known to be only associated with pathogenicity in plants, was also present in all clinical strains (Delétoile et al. 2009). Therefore, it was concluded that clinical and plant-associated strains do not form distinct populations. Altogether, the study indicated that MLSA is a powerful tool for Pantoea species delineation and identification and for strain tracking (Delétoile et al. 2009).

Of the six housekeeping genes previously described, an in-depth comparison of tree topologies derived from single genes indicates that the tree topology for the gene leuS, that encodes leucyl-tRNA synthetase, is similar to the topology produced from the concatenation of previously mentioned genes (Tambong et al.  2014). This gene may be a strong phylogenetic marker for Pantoea due to low recombination frequency and stable polymorphic sites within the coding sequence (Tambong et al. 2014).

Three primer sets are available for the detection of P. ananatis based on the 16S-23S ribosomal internal transcribed spacer (ITS) region. Although the primers were designed from the 16S-23S ITS region of P. ananatis, they cross-reacted with P. allii and P. stewartii subsp. indologenes (Carr et al. 2010; Figueiredo and Paccola-Meirelles 2012; Gitaitis et al. 2002). Asselin et al. (2016) developed PCR primers for detection of P. ananatis, Burkholderia sp., and Enterobacter sp. from onion which is specific to P. ananatis and, to date, they have not cross-reacted with other closely related Pantoea species. PCR primers specific for P. agglomerans are not available and the detection relies on 16S rRNA sequencing and MLSA. Two sets of P. stewartii subsp. indologenes specific primers are available based on recA and galE protein-coding genes that are routinely used for detection (Gehring et al. 2014).

Disease cycle and epidemiology

The life cycle of P. ananatis is complex, as bacteria can overseason to infect onions in a number of different ways (Fig. 2).  Like many bacterial pathogens, P. ananatis can be seed-borne with infested seed serving as a survival mechanism as well as a mea​ns of dissemination (Walcott et al. 2002). Walcott et al. demonstrated that P. ananatis can be both naturally seed-borne and seed-transmitted in onion. The authors collected seeds from umbels of onion plants with no obvious symptoms. Using an immunomagnetic separation and polymerase chain reaction (IMS-PCR) assay with species-specific primers, P. ananatis was detected in naturally infested onion seed. Although P. ananatis did not affect germination rates, the pathogen did cause necrosis on germinating seedlings. The significance of the bacterium's ability to colonize seed is uncertain, as most onion seed production sites are located in arid climates. Nonetheless, understanding P. ananatis' ability to infest seeds and deciphering its mechanism of seedling transmission are critical to understand center rot epidemiology (Walcott et al. 2002).

​​​​​​​​​​​​
Fig. 2. Center rot-onion pathosystem: Multiple sources of inoculum (seed, weed and thrips) contribute to center rot epidemic in field; A: foliar symptom resulting in reduced yield (pre-harvest) and B: bulb rot in storage resulting in post-harvest losses

Although P. ananatis can be seedborne, the proposed primary mode of transmission is by two insect vectors (Gitaitis et al. 2003; Wells et al. 2002). Two species of thrips, tobacco thrips (Frankliniella fusca (Hinds)) and onion thrips (Thrips tabaci), have the ability to transiently acquire and transmit P. ananatis and P. agglomerans (Gitaitis et al. 2003; Dutta et al. 2014), which are prevalent in the onion-producing region of Georgia specifically Vidalia onion region. The Vidalia onion region comprises of 21 counties in the southeastern part of Georgia, where specialty sweet Vidalia onions are grown. Immunolabeling with P. ananatis-specific antibody indicated that P. ananatis was localized in the gut of T. tabaci (Fig. 3).  Further investigation revealed that P. ananatis could not be detected in salivary secretions of T. tabaci despite feeding on a contaminated food source, but rather detected from fecal rinsates. The authors proposed that simultaneous feeding and defecating may facilitate bacterial ingress through feeding wounds from feces. The bacterium can persist in a non-circulative manner in the gut of thrips for 128 h, allowing the vector to infect plants over an extended period of time (Dutta et al. 2014).

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​Fig. 3. Immunolocalization of Pantoea ananatis in adult Thrips tabaci. (Top) Longitudinal section stained with toluidine blue of Thrips tabaci that had fed on epiphytic populations of Pantoea ananatis; (Bottom) Same section as above using fluorescent microscopy to highlight P. ananatis cells labeled with a fluorescent antibody. Mg1 = midgut 1, Mg2 = midgut 2, Mg3 = midgut 3, and Hg = hindgut.

Further studies were conducted to investigate interactions of F. fusca and P. ananatis. Both adults and larvae of F. fusca were able to acquire the bacterium, which further persisted transstadially. Moreover, both thrips' life stages were able to acquire P. ananatis within one hour of access to a contaminated food source, and by 48 h approximately 70% of thrips acquired the pathogen (Dutta et al. 2016). Mechanical inoculation of fecal rinsates on onion showed P. ananatis was able to infect tissue, while sucrose solution potentially containing salivary secretions were not able to cause symptoms suggesting the bacterium is not circulative. These observations support the hypothesis that P. ananatis infection occurs from defecation near wounding sites (Dutta et al. 2016).     

P. ananatis can survive epiphytically and endophytically on a wide range of hosts. These alternative hosts can serve as a source of inoculum in fields where susceptible crops are grown. In Georgia alone, 25 weed species, including carpetweed (Mollugo verticillata), common ragweed (Ambrosia artemisiifolia), crabgrass (Digitaria sanguinalis), common cocklebur (Xanthium pensylvanicum), curly dock (Rumex crispus), Florida pusley (Richardia scabra), sicklepod (Cassia obtusifolia), stinkweed (Thlaspi arvense), Texas panicum (Panicum texanum), vaseygrass (Paspalum urvillei), wild radish (Brassica spp.), yellow nutsedge (Cyperus esculentus) and other multiple crop plants were found to harbor P. ananatis populations asymptomatically (Gitaitis et al. 2002). Many of these weeds were found on or nearby onion production fields providing a local inoculum source (Gitaitis et al. 2002).

Of the weeds P. ananatis was found to survive consistently on Florida pusley (R. scabra) (Gitaitis et al. 2002). Dutta et al. (2017) investigated how P. ananatis survived epiphytically under different temperature and moisture regimes that mimicked conditions in the Vidalia onion region from March to May. P. ananatis populations survived significantly better at 21.1°C than 15.5°C under different moisture regimes (12 h wet/12 h dry; 12 h dry/12 h wet; continuous wet) (Dutta et al. 2017). Bacterial colonies were recovered from leaves up to 72 hours post-inoculation. However, epiphytic survival was not observed under a continuous dry regime. P. ananatis epiphytic populations survived longer on R. scabra leaves with prolonged leaf wetness at a temperature common from March to May in the Vidalia onion region. Therefore, weeds near onion production sites may harbor epiphytic bacterial populations for prolonged periods when onions are nearing maturity (Dutta et al. 2017).

Disease management

Onion cultivars resistant to Pantoea sp. are not commercially available. Use of certified onion seeds is encouraged to avoid introduction of Pantoea sp. inoculum in the production field. Planting early maturing or mid-maturing onion varieties are often recommended for growers. Late maturing varieties provide a larger window for infection and a potential epidemic to occur, which are favored by thrips pressure, hot and humid conditions, and lack of effective bactericides. These conditions are more prevalent in the southeastern United States compared to other onion growing regions of country. Early maturating varieties are able to avoid conditions that are suitable for bacterial disease development and P. ananatis infection. The overhead irrigation should be avoided as it promotes bacterial spread compared with sub-surface or drip-irrigation. Controlling thrips population can be an effective management strategy to reduce center rot incidence as these vectors play an important role in bacterial transmission (Dutta et al., 2014, 2016). An integrated approach that targets sources of inoculum to counter bacterial spread is desirable for center rot management.

Center rot management in onion fields relies heavily on copper applications mixed with an ethylenebisdithiocarbamate fungicide (EBDC), such as mancozeb, which growers may apply weekly as a protectant (Pfeufer et al. 2015). In addition, researchers found P. ananatis strains to be copper-tolerant indicating overuse and potential risk of insensitivity to this chemistry (Nischwitz et al. 2007).  It has been suggested that in other Enterobacteriales members  such as Escherichia coli and Enterococcus hirae, tolerance to copper is mediated by an efflux system, which actively expels the copper ions out of bacterial cell (Weiner et al., 1999). Another mechanism suggests the role of 'cop operon' involving periplasmic, inner and outer membrane protein coding genes that sequester copper ion in the periplasm and outer membrane (Cooksey 1990, 1994). However, no such mechanism has yet been confirmed in P. ananatis. Repeated applications of copper sprays during susceptible growth stages can be effective only to a limited extent and does not offer a robust solution to the problem. Perhaps the inefficacy of these sprays could be due to thrips preference to colonize certain parts of the onion plant, e.g. the basal meristems (neck region). Applications of copper and insecticide may not come in contact with the basal meristem leaving it susceptible to infection (Gitaitis et al. 2003). A cultivar bred with a wide angle of divergence of the innermost leaves, and leaf sheaths elongating beyond the older leaves may prevent thrips from colonizing this area (Boivin et al. 1995; Brewster 1987, 2008).

The implementation of successful weed management strategies are important in reducing P. ananatis inoculum in the field. By reducing weeds, growers​ can potentially reduce initital inoculum. For example, effective weed management has been observed to reduce the spread of Pseudomonas viridiflava in onion farmscapes located in Georgia (Gitaitis et al. 2004). ​

Genome architecture of P. ananatis

The P. ananatis genome ranges from 4.3 Mb (P. ananatis S6) to 5.25 Mb (P. ananatis DAR 76143) suggesting substantial strain-to-strain variation in gene content. P. ananatis genomes are predicted to encode over 4000 genes (Stuart et al. 2017). The genomes of 32 publicly released P. ananatis strains consist of one large circular chromosome and the large Pantoea plasmid (LPP-1) in addition to variable accessory plasmids (Ismail et al. 2014). The LPP-1 plasmid ranges in size from 280.8 to 352.8 Kb and typically encodes 200-300 predicted genes (Choi et al. 2012; Weller-Stuart et al. 2014). P. ananatis genome sequencing efforts have focused on phytopathogenic strains known to infect plant hosts including pineapple (Adam et al. 2014), rice (Choi et al. 2012), eucalyptus (De Maayer et al. 2012), cotton and maize (Medrano and Bell, 2015, Weller-Stuart et al. 2014) as well as saprophytic and plant growth promoting strains (De Maayer et al. 2014). A collection of 10 Georgia P. ananatis isolates including six onion pathogenic and four non-pathogenic strains were sequenced to identify genes related to onion pathogenicity in P. ananatis (Stice et al. 2018).

Variation in pathogenicity and aggressiveness has been linked to variations in the mobile accessory genome. The LPP-1 plasmid has been proposed to make major contributions to P. ananatis genomic plasticity and phenotypic adaptability (De Maayer et al. 2012). P. ananatis LPP-1 is derived from a plasmid ancestral to the Pantoea genus and has undergone extensive diversification in terms of its genetic composition. Several phage-associated regions and mobile genetic elements have been accumulated in the plasmid genome during the course of its evolution. Many of the plasmid encoded genes such as antibiotic resistance, iron and nitrogen assimilation, and host-microbe interaction determinants are proposed to contribute to the phenotypic diversity of this bacterium (De Maayer et al. 2012). An integrative and conjugative element (ICEPan) has also been proposed to contribute to phenotypic diversity. The ICEPan element encodes for proteins involved in antibiosis and stress responses which may contribute to the occupation of diverse niches (De Maayer et al. 2015). A recent study by Stice et al. (2018) identified 57 genes in four contiguous blocks that correlated with the ability of P. ananatis to cause both foliar and bulb symptoms in onion. These regions were named Onion Virulence Regions A-D (OVRA-D) and are carried on an accessory plasmid. The OVR loci contain genes annotated as involved in thiol and redox regulation, sugar transporters as well as pectate lyase and rhamnogalacturonase cell wall degrading enzymes.​

Virulence Factors

P. ananatis is unusual among phytopathogenic bacteria in that it does not encode a virulence-associated Type III secretion system (T3SS), a common pathogenicity determinant among other bacterial plant pathogens including P. stewartii subsp. stewartii,  nor a Type II secretion system (T2SS) used by many soft rot pathogens to secrete cell wall degrading enzymes (Stice et al. 2018). P. ananatis encodes several Type VI secretion system (T6SS) loci some of which have been shown to contribute to inter-bacterial competition and symptom development in onion leaves (Shyntum et al. 2014). Other factors identified with critical roles in onion center rot include the EanI/EanR N-acyl homoserine lactone quorum regulation system (Morohoshi et al. 2007) and flagellar swimming motility (Weller-Stuart et al. 2017). Twitching motility was also observed to make a modest contribution to disease (Weller-Stuart et al. 2017). In addition, P. ananatis encodes the gene synthesis cluster for 'stewartan' exopolysaccharide production, a critical virulence factor for P. stewartii subsp. stewartii (Stice et al. 2018). Interestingly, both flagellar gene expression and EPS production are quorum regulated in P. ananatis (Morohoshi et al. 2007; Sibanda et al. 2018). A recent study by Asselin et al. (2018) identified a chromosomal cluster of 16 genes that is strongly associated with virulence of P. ananatis in onion. This gene cluster, called HiVir, has a reduced GC content, relative to the balance of the genome, indicating its potential acquisition via horizontal gene transfer (Fig. 4). Inactivation of the Major Facilitator Family exporter gene (HiVir gene K) was associated with reduced lesion length on onion leaves. The HiVir cluster also contains pepM, a gene annotated as phosphoenolpyruvate phosphomutase (Fig. 4). Deletion of the pepM gene (HiVir gene C) is associated with the loss of symptom production in both onion leaves and bulbs, as well as a large reduction in bacterial multiplication. PepM is commonly required for the initial step in the biosynthetic pathways for phosphonates and phosphinates. The authors proposed that the HiVir cluster is required for the production of an as of yet unidentified phosphonate or phosphinate secondary metabolite that plays a major role in the pathogenicity of P. ananatis in onion (Asselin et al. 2018).​

​Fig 4. Diagram of the proposed HiVir gene cluster region from P. ananatis LMG 20103(PANA_3292- PANA_3277). Onion plants 5 days after inoculation with P. ananatis OC5a Rpr wild type (WT) and derivatives. A, WT; B, ΔpepM mutant; C, ΔpepM complemented in trans; D, ΔpepM harboring empty vector. (Reproduced from Asselin et al. 2018)

Significance

Center rot has emerged as a chronic problem in a number of onion growing regions in the United States, and has been responsible for significant pre- and post-harvest losses in yield and quality. Like many bacterial pathogens, P. ananatis can be seedborne with infested seed serving as a survival mechanism as well as a means of dissemination (Walcott et al. 2003). Also, as with many bacteria, these pathogens can survive on several weed species (Gitaitis et al. 2002). Finally, P. ananatis can be transmitted by tobacco and onion thrips making it the only plant pathogenic bacterial pathogen currently known to be thrips transmitted (Gitaitis et al. 2003; Dutta et al. 2014, 2016). Host resistance against these pathogens has not yet been characterized and onion varieties with resistance/tolerance to Pantoea spp. has not been identified. The multiple sources of inoculum (seed, weeds, and insects) and a lack of genetic resistance to P. ananatis requires evaluation of mitigation strategies for each inoculum source and ultimately a comprehensive, resilient disease management strategy against this pathogen. Breeding for host resistance will be critical for future management of this disease. For disease management and resistance breeding to be effective, an understanding of virulence mechanisms and the pathogen population structure is necessary. Representative strain(s) of taxa within the pathogen population should be considered when screening germplasms, because a diverse pathogen population may overcome resistance rather quickly, or the host may not express complete resistance. On the contrary, a population in a specific geographic region lacking diversity may be less challenging to breed resistance against P. ananatis. Hence, the future research should focus on understanding virulence mechanisms and bacterial population structure and strategies to develop advanced breeding lines that are resistant to Pantoea sp.​

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