Acidovorax citrulli (fruit blotch)
- Taxonomic Tree
- Notes on Taxonomy and Nomenclature
- Distribution Table
- Risk of Introduction
- Hosts/Species Affected
- Growth Stages
- List of Symptoms/Signs
- Biology and Ecology
- Seedborne Aspects
- Plant Trade
- Detection and Inspection
- Similarities to Other Species/Conditions
- Prevention and Control
- Distribution Maps
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IdentityTop of page
Preferred Scientific Name
- Acidovorax citrulli (Schaad et al.) Schaad et al.
Preferred Common Name
- fruit blotch
Other Scientific Names
- Acidovorax avenae subsp. citrulli (Schaad et al., 1978) Willems et al., 1992
- Pseudomonas avenae subsp. citrulli (Schaad et al., 1978) Hu et al., 1991
- Pseudomonas pseudoalcaligenes subsp. citrulli Schaad et al., 1978
International Common Names
- English: bacterial fruit blotch; seedling blight
Local Common Names
- UK: fruit rot
- PSDMAC (Acidovorax avenae subsp. citrulli)
Taxonomic TreeTop of page
- Domain: Bacteria
- Phylum: Proteobacteria
- Class: Betaproteobacteria
- Order: Burkholderiales
- Family: Comamonadaceae
- Genus: Acidovorax
- Species: Acidovorax citrulli
Notes on Taxonomy and NomenclatureTop of page
The bacterium was originally isolated from water-soaked lesions on cotyledons of watermelon seedlings from accessions in the USA plant introduction (PIs) collection (Webb and Goth, 1965). The bacterium was phenotypically similar to Pseudomonas pseudoalcaligenes but differed in that it was pathogenic to watermelon, cantaloupe, cucumber and squash (Schaad et al., 1978). Therefore, this new bacterium was named P. pseudoalcaligenes subsp. citrulli. Hu et al. (1991) found a close similarity between this bacterium and Pseudomonas avenae and thus was renamed P. avenae subsp. citrulli. The watermelon bacterium and other subspecies of P. avenae constitute a separate rRNA branch within the family Comamonadaceae. Comparing them phenotypically, Willems et al. (1992) found the members of this rRNA branch to be most closely related to the genus Acidovorax and the watermelon fruit blotch bacterium was renamed Acidovorax avenae subsp. citrulli. The current preferred name for this pathogen is Acidovorax citrulli.
DescriptionTop of page
A. citrulli is Gram-negative, obligately aerobic, and motile with a single polar flagellum (Willems et al., 1992). Cells are straight to slightly curved rods that are 0.2 to 0.8 by 1.0 to 5.0 um. On nutrient agar, colonies are round with slightly scalloped or spreading margins. Colonies are convex, smooth to slightly granular, and beige to faintly yellow with a translucent marginal zone. Colonies are non-fluorescent on King's medium B.
DistributionTop of page
A. citrulli has been distributed throughout the watermelon growing areas of the USA on contaminated seed, and probably in many other watermelon producing areas of the world (Latin and Hopkins, 1995). The geographical origin of the bacterium is not known. The bacterium was detected in the USA plant introduction collection several years before it appeared in commercial watermelon (Webb and Goth, 1965).
A. citrulli was detected in watermelons grown in Tipitapa in Nicaragua in 1997 from contaminated seed (Munoz and Monterroso, 2002). In 2004 CABI was informed by the Ministerio Agropecuario y Forestal, Nicaragua, that the disease was eradicated and the pathogen had not been detected in subsequent plant and soil tests of the area.
Distribution TableTop of page
The distribution in this summary table is based on all the information available. When several references are cited, they may give conflicting information on the status. Further details may be available for individual references in the Distribution Table Details section which can be selected by going to Generate Report.
|Continent/Country/Region||Distribution||Last Reported||Origin||First Reported||Invasive||Reference||Notes|
|China||Present||CABI/EPPO, 2011; EPPO, 2014|
|-Anhui||Present||Tian et al., 2013; EPPO, 2014|
|-Fujian||Present||Cai et al., 2005; CABI/EPPO, 2011; EPPO, 2014|
|-Hainan||Present||CABI/EPPO, 2011; EPPO, 2014|
|-Heilongjiang||Present||Wang et al., 2013|
|-Jilin||Present||Jin et al., 2004; CABI/EPPO, 2011; EPPO, 2014|
|-Nei Menggu||Present||Zhao et al., 2001; CABI/EPPO, 2011; EPPO, 2014|
|-Xinjiang||Present||Zhao et al., 2001; CABI/EPPO, 2011; EPPO, 2014|
|-Yunnan||Present||Zhu et al., 2013; EPPO, 2014|
|Indonesia||Absent, unreliable record||EPPO, 2014|
|Iran||Absent, unreliable record||Harighi, 2007; CABI/EPPO, 2011; EPPO, 2014|
|Israel||Present, few occurrences||Burdman et al., 2005; CABI/EPPO, 2011; EPPO, 2014|
|Israel||Transient: actionable, under eradication||Burdman et al., 2005; CABI/EPPO, 2011; EPPO, 2014|
|Japan||Present||Ogiso et al., 2001; CABI/EPPO, 2011; EPPO, 2014|
|-Honshu||Present||CABI/EPPO, 2011; EPPO, 2014|
|Korea, Republic of||Present||CABI/EPPO, 2011; EPPO, 2014|
|Malaysia||Present||CABI/EPPO, 2011; EPPO, 2014|
|Taiwan||Present||Wang and Cheng, 2001; CABI/EPPO, 2011; EPPO, 2014|
|Thailand||Present||CABI/EPPO, 2011; EPPO, 2014|
|Turkey||Present||Demir, 1996; CABI/EPPO, 2011; EPPO, 2014|
|USA||Restricted distribution||CABI/EPPO, 2011; EPPO, 2014|
|-Alabama||Present, few occurrences||Hopkins et al., 1994; CABI/EPPO, 2011; EPPO, 2014|
|-Arkansas||Present, few occurrences||Hopkins et al., 1994; CABI/EPPO, 2011; EPPO, 2014|
|-California||Present, few occurrences||Hopkins et al., 1994; CABI/EPPO, 2011; EPPO, 2014; Kumagai et al., 2014|
|-Delaware||Absent, formerly present||Evans and Mulrooney, 1991; CABI/EPPO, 2011; EPPO, 2014|
|-Florida||Present||Somodi et al., 1991; CABI/EPPO, 2011; EPPO, 2014|
|-Georgia||Present||Hopkins et al., 1994; Walcott et al., 2000; CABI/EPPO, 2011; EPPO, 2014|
|-Illinois||Present||Babadoost and Pataky, 2002; CABI/EPPO, 2011; EPPO, 2014|
|-Indiana||Present, few occurrences||Latin and Rane, 1990; CABI/EPPO, 2011; EPPO, 2014|
|-Iowa||Present||Hopkins et al., 1994; CABI/EPPO, 2011; EPPO, 2014|
|-Maryland||Present||Hopkins et al., 1994; CABI/EPPO, 2011; EPPO, 2014|
|-Mississippi||Present, few occurrences||Hopkins et al., 1994; CABI/EPPO, 2011; EPPO, 2014|
|-Missouri||Present, few occurrences||Hopkins et al., 1994; CABI/EPPO, 2011; EPPO, 2014|
|-North Carolina||Present, few occurrences||Hopkins et al., 1994; CABI/EPPO, 2011; EPPO, 2014|
|-Oklahoma||Present, few occurrences||Jacobs et al., 1992; CABI/EPPO, 2011; EPPO, 2014|
|-Oregon||Present, few occurrences||Hamm and Spink, 1997; CABI/EPPO, 2011; EPPO, 2014|
|-South Carolina||Present||Hopkins et al., 1994; CABI/EPPO, 2011; EPPO, 2014|
|-Texas||Present||Black et al., 1994; CABI/EPPO, 2011; EPPO, 2014|
Central America and Caribbean
|Costa Rica||Present||Mora-Umaña and Araya, 2002; CABI/EPPO, 2011; EPPO, 2014|
|Nicaragua||Eradicated||Munoz and Monterroso, 2002; CABI/EPPO, 2011; EPPO, 2014|
|Trinidad and Tobago||Present||EPPO, 2014|
|Brazil||Present||CABI/EPPO, 2011; EPPO, 2014|
|-Ceara||Present||Sales et al., 2005; CABI/EPPO, 2011; EPPO, 2014|
|-Minas Gerais||Present||CABI/EPPO, 2011; EPPO, 2014|
|-Pernambuco||Present||CABI/EPPO, 2011; EPPO, 2014|
|-Rio Grande do Norte||Present||Assis et al., 1999; CABI/EPPO, 2011; EPPO, 2014|
|-Rio Grande do Sul||Present||CABI/EPPO, 2011; EPPO, 2014|
|-Roraima||Present||Halfeld-Vieira and Nechet, 2007; CABI/EPPO, 2011; EPPO, 2014|
|Greece||Restricted distribution||CABI/EPPO, 2011; EPPO, 2014|
|-Greece (mainland)||Restricted distribution||CABI/EPPO, 2011|
|Hungary||Present, few occurrences||Palkovics et al., 2008; CABI/EPPO, 2011; EPPO, 2014|
|Italy||Present, few occurrences||CABI/EPPO, 2011; EPPO, 2014|
|-Italy (mainland)||Present, few occurrences||CABI/EPPO, 2011|
|-Sardinia||Transient: actionable, under eradication||EPPO, 2014|
|Netherlands||Absent, confirmed by survey||NPPO of the Netherlands, 2013; EPPO, 2014|
|Australia||Present, few occurrences||CABI/EPPO, 2011; EPPO, 2014|
|-Queensland||Present, few occurrences||Queensland Dept. Primary Ind., 1978; O'Brien and Martin, 1999; Martin and Horlock, 2002; CABI/EPPO, 2011; EPPO, 2014|
|Guam||Present||Wall and Santos, 1988; Wall et al., 1990; CABI/EPPO, 2011; EPPO, 2014|
|Northern Mariana Islands||Present||Wall and Santos, 1988; Wall et al., 1990; CABI/EPPO, 2011; EPPO, 2014|
Risk of IntroductionTop of page
There are no known quarantine restrictions on A. citrulli.
Hosts/Species AffectedTop of page
Symptoms can be produced in all cucurbits tested by inoculation, especially in the seedling stage (Schaad et al., 1978; Latin and Hopkins, 1995). Watermelon, cantaloupe and honeydew melons appear the most susceptible, with both foliar symptoms and blotch symptoms on the fruit (Isakeit et al., 1997). Symptoms develop on inoculated foliage of squash, cucumbers and other cucurbits, but fruit symptoms have not been observed on these hosts. Citron (Citrullus lanatus var. citroides), a common weed in parts of the southern USA, is also a host for A. citrulli. Symptoms are produced on the foliage and fruit, and seed transmission occurs in this cucurbit weed, giving it the potential to serve as an alternate host to perpetuate the bacterium.
In host range studies, symptoms were produced on tomato, eggplant and pepper foliage, but not on fruit.
Growth StagesTop of page Fruiting stage, Seedling stage, Vegetative growing stage
SymptomsTop of page
Initial symptoms of A. citrulli in watermelon seedlings appear as water-soaked areas on the underside of cotyledons and leaves (Webb and Goth, 1965). In young seedlings, lesions can develop in the hypocotyl resulting in collapse and death of the emerging plant. As cotyledons expand, water-soaked lesions turn dark brown and often extend along the length of the midrib. Leaf lesions are light brown to reddish-brown and frequently spread along the midrib of the leaf (Latin and Hopkins, 1995). As the growing season progresses, leaf symptoms may become sparse and inconspicuous.
The characteristic symptom of bacterial fruit blotch in watermelon is a dark, olive-green blotch on the upper surface of infected fruit that begins as a small, water-soaked area a few millimetres in diameter and rapidly enlarges to a lesion several centimetres in diameter with irregular margins (Somodi et al., 1991). In a few days, the lesions may expand to cover the entire upper surface of the fruit , leaving only the groundspot symptomless. Initially, the lesions do not extend into the flesh of the watermelon. In advanced stages of lesion development, the initial infection site may become necrotic. Cracks in the rind surface may occur, resulting in fruit rot. Rotting watermelon fruit often ooze a sticky, clear, amber substance or an effervescent exudate (Latin and Hopkins, 1995).
Seedling and leaf symptoms on other cucurbits are similar to those on watermelon. Symptoms on muskmelon fruit consist of water-soaked pits on the fruit surface (Latin and Hopkins, 1995). In honeydew fruit, lesions begin as water soaked spots that, with age, become brown and cracked in the centre with a water-soaked margin. The lesions in honeydew are usually 3-10 mm in diameter.
List of Symptoms/SignsTop of page
|Fruit / lesions: black or brown|
|Leaves / necrotic areas|
|Whole plant / seedling blight|
Biology and EcologyTop of page
The disease cycle in most host crops apparently begins with contaminated seed (Latin and Hopkins, 1995). A. citrulli can survive for several years on seeds that have been dried and stored. Much of the watermelon production in the USA is still from direct-seeded plants, but transplants are rapidly gaining in popularity. A warm, humid environment (favourable for disease development) is maintained in transplant facilities used for raising watermelon seedlings. Bacteria from infested seed infect the developing seedling as the cotyledons emerge from the seed coat. Most transplant facilities employ overhead irrigation, which effectively splash-disperses bacteria to neighbouring seedlings. This secondary spread in the transplant house can result in high numbers of infected seedlings reaching the field. Some of these transplants may harbour the bacterium, but show no symptoms. Because a single transplant house may supply plants to several growers over a large geographical area, transplants can be the mechanism through which the bacterium is dispersed.
The fruit blotch bacterium may be introduced into a field from infested seed, infected transplants, contaminated volunteer crop cucurbits, or infected wild cucurbits. In the field, symptom development and spread of A. avenae subsp. citrulli on foliage and fruit is most rapid during periods when the weather is hot and humid, with thundershowers (Hopkins, 1993). These conditions are common during the summer. In addition to wind-driven rain, the bacterium can be spread by contact with farm workers, irrigation, and other cultivation equipment (D. Hopkins, University of Florida, USA, personal communication, 1997). The bacterium does not appear to spread as rapidly during cool, rainy weather. With favourable weather, a few primary infection sites in a field can result in infection of all plants by the time of harvest. Leaf lesions in the field do not result in defoliation, but are important reservoirs of bacteria for fruit infection. Under some environmental conditions, foliar symptoms may not be very conspicuous and the watermelon grower may not realize that there is a problem until fruit symptoms render the crop unmarketable.
A. citrulli is spread from leaf lesions to developing fruit by splash dispersal during rain or irrigation. Watermelon fruits are most vulnerable to bacterial infection 2 to 3 weeks after anthesis (Frankle et al., 1993). The bacterium invades the fruit through stomata on the fruit surface. Small, water-soaked lesions develop 3-7 days later. Mature watermelon fruit are covered with a wax layer that plugs stomata and prevents the entry of bacteria into the fruit. Once the wax layer forms, mature watermelons can be invaded by the bacterium only after wounding. Thus, the bacterium must invade the fruit seven days, or more, before maturity, but the symptoms continue to develop as long as the fruit is attached to the plant. Surface lesions cease to enlarge after harvest.
Wild cucurbits can become infected with A. citrulli by spread from cucurbit crops. Volunteer cucurbits that grow from previous crops that had fruit blotch can be naturally infected with the bacterium. These infected wild cucurbits and volunteer crop cucurbits may be important overseasoning hosts for the bacterium that helps to perpetuate the disease in an area (Latin and Hopkins, 1995). Production of cucurbit crop seed in areas with these alternate hosts of A. citrulli can result in the production of infested seed that begin the disease cycle again.
Seedborne AspectsTop of page
There are very few reports of the incidence of A. citrulli in commercial seed lots. However, the infection level generally has been low, because in heavily infected seed lots the bacterium would have been detected and seed would not have been sold. One commercial seed lot was reported, in a grow-out test, to have one infected seed in 9000 (Rane and Latin, 1992). Sowell and Schaad (1979) reported disease incidence on USA plant introductions of watermelons to be over 50% in some cases, however this was based on only 25 seeds. Seed samples collected from symptomatic fruit have had infection levels over 80% (Kucharek et al., 1993).
Walcott et al. (2003) investigated the role of watermelon blossom inoculation in seed infestation by A. citrulli. Approximately 98% (84/87) of fruit developed from blossoms inoculated with 1 x 107 or 1 x 109 c.f.u. of A. citrulli per blossom were asymptomatic. Using immunomagnetic separation and PCR, A. avenae subsp. citrulli was detected in 44% of the seed lots assayed, despite the lack of fruit symptoms. Furthermore, viable colonies were recovered from 31% of the seed lots. Of these lots, 27% also yielded seedlings expressing bacterial fruit blotch symptoms when planted under conditions of 30°C and 90% RH. A. citrulli was detected and recovered from the pulp of 33 and 19%, respectively, of symptomless fruit whose blossoms were inoculated with A. citrulli. The ability to penetrate watermelon flowers was not unique to A. citrulli, because blossoms inoculated with Pantoea ananatis also resulted in infested seed and pulp. The data indicate that watermelon blossoms are a potential site of ingress for fruit and seed infestation by A. citrulli. These data indicate that watermelon blossoms are a potential site of ingress for fruit and seed infestation by A. citrulli. Lessl et al. (2007) reported that A. citrulli can rapidly colonize watermelon stigmas reaching populations of 1 x 109 c.f.u/ blossom by 96 h post-inoculation. The authors also showed that low levels of A. citrulli (1 x 103 c.f.u /blossom) applied to stigmas can lead to seed infestation. Subsequently, using a constitutive green fluorescent protein mutant of A. citrulli, it was demonstrated that the bacterium can penetrate the stigma and style via the transmitting tract tissues and reach the ovary by 7 days after inoculation (Lessl, 2003). This mechanism of seed infestation provides a possible explanation for the production of contaminated seedlots from fields with no visible BFB symptoms. In addition, recently, it was reported that blossom or stigma inoculation resulted primarily in bacterial localization in the seed embryo, whereas pericarp inoculation resulted in bacterial deposition under the seed coat (Dutta et al., 2012). These aspects may have implications on bacterial survival in seed and efficiency of pathogen detection by seed health testing.
Effect on Seed Quality
There does not appear to be any effect of A. citrulli on seed quality. Seed appearance, germination and seedling vigour are unaffected.
The 1989 outbreak of bacterial fruit blotch of watermelon in the USA appeared to be initiated by contaminated seed. Disease symptoms appeared in Florida, South Carolina, North Carolina, Maryland, Delaware and Indiana, all within a few months. In Indiana, initial symptoms in diseased seedlings in commercial greenhouses were limited to distinct foci (Rane and Latin, 1992). Many of the fruit blotch infected greenhouses and fields were associated with a seed lot that was later shown to be infested with A. citrulli. Laboratory and greenhouse tests have demonstrated transmission of the bacterium from infested seeds to as high as 80% of the seedlings from these seeds (Kucharek et al., 1993; Hopkins et al., 1996). Other studies have corroborated transmission of the pathogens by seeds. A. citrulli was detected in 44% of the seed lots assayed using immunomagnetic separation and PCR. Viable colonies were recovered from 31% of the seed lots. Of these lots, 27% also yielded seedlings expressing bacterial fruit blotch symptoms when planted under conditions of 30°C and 90% RH (Walcott et al., 2003). Walcott et al. (2000) showed that A. citrulli can infest seeds within symptomless watermelon fruits, by inoculation through the stigma of female blossom. Seedlots generated by stigma-inoculation yielded seedlings expressing BFB symptoms when planted under conditions of 30°C and 90% RH (Walcott et al., 2003). In Australia, tests showed A. citrulli was readily seed transmissible from naturally infected rockmelon and honeydew fruit for at least 3 months after seed extraction (O'Brien and Martin, 1999).
In a recent study, it was reported that A. citrulli transmission as well as spatial and temporal spread in the greenhouse depends on the inoculum concentration in seed (Dutta et al., 2012). Researchers observed a sigmoidal relationship between A. citrulli seed inoculum concentration and frequency of bacterial fruit blotch (BFB) seedling transmission. One-hundred and 96.6% of seedlots containing one seed with 1 ×107 and 1 ×105 c.f.u of A. citrulli, respectively, transmitted the pathogen to seedlings; in contrast, BFB seedling transmission were lower for seedlots with one seed infested with 1 × 103 (46.6%) and 1 × 101 c.f.u of A. citrulli (16.7%). As disease transmission was observed for seedlots with just one seed containing as few as 10 c.f.u of A. citrulli, zero tolerance for seedborne A. citrulli is recommended for effective BFB management.
Furthermore, it was also observed that under greenhouse conditions without typical automated overhead irrigation, BFB developed faster from seedlots with one seed infested with ≥1 × 105 c.f.u than from seedlots with one seed infested with ≤1 × 103 c.f.u. It was also demonstrated an increase in spatial spread of BFB in the greenhouse with increase in A. citrulli seed inoculum concentration. Lots with one seed with high inoculum loads (1 ×107 or 1 × 105 A. citrulli c.f.u) led to higher frequency of disease spread compared to lots with lower levels of inoculum (1 × 103 or 1 × 101 c.f.u).
Infested seed is the primary way in which A. citrulli has been introduced into the transplant house or into watermelon fields (D. Hopkins, University of Florida, USA, personal communication, 1997). Alternative hosts can also serve as the source of inoculum into cucurbit fields. The conditions in a transplant house are highly conducive for BFB development (Walcott, 2008). Infested seeds are normally the primary source of inoculum, and secondary spread of bacteria is due to the splash-dispersal facilitated by overhead irrigation that generates bacterial aerosols. Bacteria landing on uninfected seedlings penetrate cotyledons and leaves via stomata and multiply rapidly in intercellular space. Numerous secondary infection cycles can take place due to splash dispersal.
Infected seeds and seedlings are the most important primary sources of inoculum in commercial fruit production field. However, there may be other endemic sources of inoculum like debris from infected fruit or foliage tissue, volunteer watermelon seedlings, or cucurbitaceous weeds (Black et al., 1994; Latin and Hopkins, 1995; Isakeit et al., 1998). In the field, BFB development is heavily dependent on rainfall and relative humidity. Secondary dispersal of A. citrulli is by wind-driven rain or over-head irrigation. When A. citrulli lands on healthy leaves, it migrates through open stomata into the sub-stomatal intercellular spaces where it multiplies and induces water-soaked lesions. Although, there is currently no evidence of systemic A. citrulli movement throughout the plant, there are reports where bacteria deposited on the surfaces of immature ovaries migrated through open stomata to initiate fruit infections (Frankle et al., 1993). This infection court is accessible for 2-3 weeks after anthesis, and as fruits mature, wax becomes deposited over the stomata preventing further bacterial entry. Despite this, A. citrulli can exist epiphytically on leaf and fruit surfaces and may invade tissues through wounds. Seeds from infected fruits may become buried in the soil where they may serve as an inoculum for subsequent crops. While fruit infection is initiated at anthesis, fruit symptoms do not develop until harvest maturity.
Alternative hosts can also serve as the source of inoculum in cucurbit fields.
Several studies have been conducted on the control of bacterial fruit blotch of watermelon with seed treatments. Sowell and Schaad (1979) reported that soaking seed known to be infested with A. citrulli in 1 mg/ml streptomycin for 16 h eliminated the infection of seedlings grown from these seed. However, this treatment has not been developed into an effective commercial treatment. Hot-water treatment of infested seed at 50°C for 20 and 30 minutes was shown to eliminate seed transmission to watermelon seedlings (Wall, 1989). In a later study, treatments of infested seed in 0.525% NaOCl for 20 minutes, in 1.8% HCl for 5 minutes, and in water at 50°C for 20 minutes, all significantly reduced the number of diseased seedlings that developed from infested seed, but none of the treatments prevented disease (Rane and Latin, 1992). Nomura and Shirakawa (2001) showed that hot-water treatment of infested seeds at 54-56°C for 10-30 minutes reduced disease incidence but did not eradicate the pathogen. They also showed that, among bactericide treatments, antibiotic preparations were more effective than copper compounds and other pesticides. Moreover, low concentration and lengthy soaking treatments were far superior to high concentration and short treatments. However, bactericide treatment under vacuum was not as good as the normal treatment. Although bactericide treatment reduced disease incidence, it did not eradicate the pathogen.
In a study of wet seed treatments applied at seed harvest, fermentation of seeds in watermelon juice and debris for 24 to 48 hours, or treatment in 1% HCl for 15 minutes, always reduced the seed transmission to less than 1% and, in most tests, eliminated it (Hopkins et al., 1996). Fermentation for 24-48 hours plus treatment with HCl was most effective in eliminating seed transmission. These treatments are effective on diploid watermelon seeds, but germination of triploid seed can be reduced by the treatments.
Fermentation of freshly harvested seed in watermelon fruit juice is a common practice routinely employed in commercial seed production. The process involves incubation of seeds in fruit juice for 24-48 h followed by rinsing and drying. Hopkins et al. (1996) demonstrated that fermentation of watermelon seed in fruit juice for 24h followed by rinsing and drying completely eliminated BFB seedling transmission without affecting seed germination. However, fermentation cannot be used for certain watermelon hybrids and other cucurbits as it can adversely affect seed germination (Walcott, 2008). Many seed treatments have been reported to reduce BFB seedling transmission. Treatments with 0.5-1% CaOCl, or NaOCl for 15-20 min reduced BFB seedling transmission but failed to eliminate the pathogen from the seeds (Hopkin, 1996; Hopkins et al., 2003). Hopkins also found that treatment of infested seeds with 1600 μg of peroxyacetic acid per litre of water for 30 min followed by drying at 40°C for 48 h completely eliminated BFB seedling transmission (Hopkins et al., 2003). Currently, wet seed treatment with peroxyacetic acid is a standard practice by cucurbit seed producers. There have been many other attempts to develop seed treatments for BFB. For example, Feng et al. (2009) evaluated cupric sulfate, acidified cupric sulfate, acidified cupric acetate (ACA), acidified zinc chloride, NaOCl, acidic electrolyzed water (AEW) and peroxyacetic acid. Seed treatments with NaOCl at 50oC for 20 min, peroxyacetic acid for 30 min and ACA at 50oC for 20 min eradicated the pathogen, seed germination was negatively affected. Among the seed treatments, only AEW eradicated A. citrulli from infested seeds without decreasing seed germination or seedling establishment
Seed Health Tests
The recommended test to detect A. citrulli is a seedling grow-out test conducted under conditions favourable for symptom development. The watermelon seed industry is currently testing their seed lots by grow-out test of 10,000 to 50,000 seedlings per seed lot. In this test, seeds are planted in a sterile potting mix in a greenhouse that is free from other sources of A. citrulli, usually a greenhouse dedicated to seed testing. Seedlings are watered by overhead irrigation to promote foci of symptomatic plants that are easily visible. Relative humidity in the greenhouse is maintained above 55% and temperature is maintained between 24 and 38°C (Hopkins, 1994; Latin et al., 1995). After 16-21 days, each seedling is carefully inspected for symptoms. Isolations are made from seedlings showing any symptoms of disease and A. citrulli is identified using biochemical, DNA and pathogenicity tests. This method takes 30-35 days to complete. There is zero tolerance for contaminated seed and any infested seed lot is rejected.
Elaborate precautions must be taken to ensure that cross-contamination of seedlings does not occur in test procedures. A detailed step-by-step seedling grow-out method that describes such precautions and is standardized by the USA National Seed Health System (NSHS) can be accessed at the website www.seedhealth.org.
PCR-Wash (Seminis Inc.)
A detailed step-by-step description of the Seminis Inc. PCR-Wash procedure, standardized by the USA National Seed Health System (NSHS) can be accessed at the website www.seedhealth.org.
Immunomagnetic separation and PCR (Walcott and Gitaitis, 2000)
An immunomagnetic separation and polymerase chain reaction (IMS-PCR)-based assay was developed for detecting A. citrulli in watermelon seed. IMS yielded a 10-fold increase in recovery of A. citrulli over direct spread-plating on King's Medium B; however, the presence of seed debris reduced IMS efficiency. Synthetic oligonucleotide primers were designed based on the 16S rRNA gene of a known A. citrulli strain and tested for specific DNA amplification by PCR. The primers amplified DNA from all A. citrulli strains tested but also yielded amplicons with several closely related bacteria. IMS-PCR resulted in a 100-fold increase in A. citrulli detection sensitivity over direct PCR and was unaffected by PCR inhibitors in watermelon seed. The threshold of A. citrulli detection for IMS-PCR was 10 c.f.u./ml in watermelon seed wash, and seed lots with 0.1% infestation were consistently detected. IMS-PCR represents an efficient and sensitive approach to detecting A. citrulli in watermelon seedlots.
To improve the efficiency of seed health testing, immunomagnetic separation combined with polymerase chain reaction (IMS-PCR) was evaluated (Walcott and Gitaitis, 2000; Walcott et al., 2006). This approach was necessary because watermelon seed extracts contain compounds that inhibit PCR, yielding false-negative results (Walcott and Gitaitis, 2000). With IMS-PCR, paramagnetic beads coated with anti- A. citrulli polyclonal antibodies specifically bind target cells. Immunomagnetic beads can then be rinsed to eliminate inhibitory compounds and non-target cells and the captured A.citrulli cells are lysed by boiling and the DNA is detected by conventional or real-time PCR. Using artificially infested watermelon seedlots (n = 10,000 seeds) with 0.01 and 0.1% infestation, detection frequencies of 25 and 87.5% respectively were reported for IMS-PCR as compared to 12.5 and 37.5% for the greenhouse seedling grow-out assay (Walcott et al., 2006). While the differences in detection frequency for IMS-PCR and seedling grow-out were not statistically significant, the data demonstrated the potential of IMS-PCR as an effective alternative for seed health testing. The threshold of A. citrulli detection for IMS-PCR was 10 c.f.u/ml in watermelon seed wash, and seed lots with 0.1% (n = 10,000 seeds) infestation were consistently detected. In a recent study, Dutta et al. (2012) observed a sigmoidal relationship between A. citrulli seed inoculum concentration and frequency of pathogen detection in infested seedlots using IMS combined with a real-time PCR assay. Whereas 100% of samples from seedlots (n = 10,000 seeds/lot) with one seed containing ≥1 × 105 c.f.u tested positive for A. citrulli , 75 and 16.67% of samples from lots with one seed containing 1 ×103 and 10 c.f.u, respectively tested positive for the pathogen.
Methods of seed detection of A. citrulli are being developed that are highly sensitive and require less time to conduct than the seedling grow-out test. A PCR method is highly sensitive and can be completed in 3 days (Minsavage et al., 1995). Other techniques for detection of the pathogen include ELISA (Wang and Cheng, 2001) and latex-flocculation (Kawano and Takahashi, 2001). However, these reports do not necessarily indicate detection of the pathogen in seeds.
Plant TradeTop of page
|Plant parts liable to carry the pest in trade/transport||Pest stages||Borne internally||Borne externally||Visibility of pest or symptoms|
|Fruits (inc. pods)||Yes||Yes||Pest or symptoms usually visible to the naked eye|
|Seedlings/Micropropagated plants||Yes||Pest or symptoms usually invisible|
|Stems (above ground)/Shoots/Trunks/Branches||Yes||Pest or symptoms usually visible to the naked eye|
|True seeds (inc. grain)||Yes||Pest or symptoms usually visible to the naked eye|
|Plant parts not known to carry the pest in trade/transport|
|Growing medium accompanying plants|
ImpactTop of page In the spring of 1989 in the USA, bacterial fruit blotch of watermelon first occurred in commercial watermelon fields in Florida and, as the season progressed, the disease was observed in South Carolina, North Carolina, Maryland, Delaware, and Indiana (Somodi et al., 1991; Latin and Rane, 1990). In some fields, losses were more than 90% of the total marketable fruit. Since then, the disease also has been found in Alabama, Arkansas, California, Georgia, Iowa, Mississippi, Missouri, Oklahoma, Oregon and Texas. Bacterial fruit blotch of watermelon has continued to be a threat to the watermelon industry in the USA. In most years, the disease has occurred in relatively few fields but has been devastating in many of these, sometimes resulting in the loss of all marketable fruit. Losses to individual growers have been over $100,000 in many of these cases (D. Hopkins, University of Florida, USA, personal communication, 1997). Fruit blotch was prevalent in Georgia in 1992 and was especially widespread in 1994, causing losses in thousands of hectares distributed over at least 10 states.
DiagnosisTop of page
Symptomatic cotyledon, leaf, or rind tissue is triturated in sterile water. A loopful of the suspension is streaked onto nutrient agar, King's medium B agar and a Tween base agar, such as TC50 (Frankle, 1992). Colonies of A. citrulli are nonfluorescent on the King's medium B agar and, because of lipolytic activity, produce a white halo after 3-4 days growth on the Tween agar. The hypersensitive response is produced in tobacco and tomato (Somodi et al., 1991). Pathogenicity tests are conducted by misting a bacterial suspension (106 CFU/ml) in water on young watermelon seedlings (first-true-leaf stage) until runoff occurs (Hopkins et al., 1993). With A. citrulli, water-soaked spots will occur on the underside of the cotyledons and first true leaf 3-5 days after inoculation. Alternatively, young watermelon seedlings can be inoculated by dipping a sterile toothpick into the bacterial colony and puncturing the watermelon hypocotyl with the toothpick. Either a water-soaked lesion will develop at the puncture site or the entire seedling will collapse and decay.
A. citrulli colonies can also be identified using the Microbial Identification System (MIS) fatty acid profile system (Somodi et al., 1991; Hodge et al., 1995) or the Biolog GN Microplates with the MicroLog data base (Isakeit et al., 1997). PCR can be used to diagnose A. citrulli with DNA extracts from bacterial colonies or from diseased tissue using the appropriate primers (Minsavage et al., 1995).
Seedlings showing BFB symptoms can also be confirmed by taking small (~4 mm2) pieces of symptomatic tissue and cutting into fine pieces in a drop of sterile water or 0.1M phosphate buffer saline. Using a sterile inoculation loop, a loopful of tissue macerate can be streaked on to appropriate semi-selective media and incubated for 2-3 days at 28°C. For further confirmation, putative A. citrulli colonies can be tested by extracting genomic DNA from isolated colonies and amplifying with a TaqMan real-time PCR assay using A. citrulli-specific primers (Dutta et al., 2012).
Apart from these methods, A. citrulli can also be confirmed by commercially available Immunostrips (Agdia Inc.). This is a rapid tool for detecting pathogen from leaves, fruit, bacterial cultures, and seedlings exhibiting BFB symptoms. Although the threshold of detection is 4 x 105 cells/ml, it is easy to conduct and does not require any special equipment or expertise.
Detection and InspectionTop of page Inspect young cucurbit seedlings for the typical water-soaked areas on the underside of cotyledons and the restricted lesions that turn dark brown and often extend along the length of the cotyledon midrib (Webb and Goth, 1965). In warm, humid environments, symptoms can be observed 3-4 days after seedling emergence. Also, inspect young seedlings for rotting (melting) of the hypocotyl, resulting in a complete collapse of the emerging plant. Inspect cucurbit transplants for small, dark brown lesions, often surrounded by a band of chlorotic tissue. Inspect the upper surface of mature fruit for the typical blotch symptoms.
Similarities to Other Species/ConditionsTop of page
The seedling and leaf symptoms caused by A. citrulli in cucurbits may be confused with angular leaf spot caused by Pseudomonas syringae pv. lachrymans. These two bacterial pathogens can be distinguished by the production of fluorescent pigments on King's medium B agar.
There is also another weakly pathogenic bacterium that produces symptoms in watermelon seedlings in the transplant house that could be confused with those produced by A. citrulli (Hodge et al., 1995). It produces restricted lesions on cotyledons in the transplant house, but does not produce symptoms in the field on watermelon leaves and fruit. It can be distinguished by fatty acid profile and malonate utilization. Based on fatty acid profiles, this weakly aggressive bacterium was most closely related to Acidovorax facilis.
Prevention and ControlTop of page
Due to the variable regulations around (de)registration of pesticides, your national list of registered pesticides or relevant authority should be consulted to determine which products are legally allowed for use in your country when considering chemical control. Pesticides should always be used in a lawful manner, consistent with the product's label.
Fruit of some cultivars are more susceptible to bacterial fruit blotch than others. The susceptibility of fruit appears to be related to rind colour. The most susceptible fruit are those with a light green rind, more tolerant are those with light and dark-green stripes and most tolerant are those with a solid dark-green rind (Hopkins et al., 1993). Triploid watermelons are less susceptible to A. citrulli than diploid watermelons (Rhodes et al., 1996). However, the level of tolerance currently available in commercial watermelon cultivars will not guarantee freedom from fruit blotch under conditions which favour disease development.
Exclusion of Pathogen
The ideal control is to prevent the introduction of A. citrulli into the field (Hopkins, 1997). Plant seeds that have been tested for the presence of the fruit blotch bacterium. A negative assay is not proof that the seedlot is free of the bacterium, but does indicate that disease was not detected in a representative sample of the seedlot. Use transplants from houses in which there were no seedling symptoms of the fruit blotch disease.
Cultural Control and Sanitary Methods
Cultural practices in the transplant house should include minimal manipulation of plants; decontamination of hands, plant containers, and tools after contact with plants; closing sides of the transplant house during storms or windy periods; and destroying discarded plant material. Spread of the bacterium in the greenhouse can be minimized by low humidity, low temperatures, and bottom watering (Hopkins, 1994; Latin et al., 1995). Irrigation with an overhead boom may result in splash dispersal of the bacteria throughout the greenhouse. Spread of the bacteria may be reduced by practices that minimize or eliminate long periods when leaves are wet. It would also be advisable to segregate different seed lots in the transplant house to reduce the chance of cross contamination. Decontaminate a transplant house that had infected seedlings and wait at least 2-3 weeks to plant cucurbits in it again. A 0.5% sodium hypochlorite solution, as well as commercial bactericides, may be used for decontamination.
The possibility of A. citrulli being introduced into the field from contaminated volunteer watermelons can be reduced by ploughing under debris, including watermelon culls, from an infected field (D. Hopkins, University of Florida, USA, personal communication, 1997). The current field should be planted as far as possible from the previous crop. Volunteer watermelon seedlings from previous crops should be destroyed immediately to eliminate potential sources of the fruit blotch bacterium. In northern growing areas, fruit blotch affected fields should be planted subsequently with crops that utilize herbicides that will kill volunteer watermelons. Eliminate wild cucurbits and volunteer cucurbits near transplant houses and production fields. Do not work in an infested field if the foliage is wet. Decontaminate irrigation or mechanical equipment before moving it from an infested field to a noninfested field.
Applications of copper-containing fungicides at the full recommended rates have reduced the incidence of fruit blotch symptoms when applications were initiated prior to fruit set (Hopkins, 1991; Hopkins, 1995). If fruit blotch symptoms are observed, applications of copper-containing fungicides should begin at first flower, or earlier, and continue weekly until all fruit are mature. As a preventive treatment, biweekly applications of the full rate of copper or weekly applications at half the recommended rate of copper-containing fungicides may be used.
ReferencesTop of page
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Distribution MapsTop of page
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