Bacterial fruit blotch of cucurbits

The seminar disclaimer applies to this post.

Seminar this week was given by Dr. Ronald Walcott from the University of Georgia (UGA), Athens. He got his BS and MS degree from Iowa State University, and his PhD from UGA. While searching the internet for references related to this post, I found this interview with Dr. Walcott. Dr. Walcott studies seed-borne plant pathogens.

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Bacterial fruit blotch (BFB) has become a significant pathogen over the years, in particular on watermelon. The first symptoms normally appear on the cotyledons, afterwards symptoms can sometimes be seen on the true leaves too. Under field conditions, it is really hard to distinguish these symptoms, because they are rather nondescript. The main problem occurs later in the season, when the fruits show irregularly shaped blotches, the fruits crack, and the insides of the fruit ooze out. Contrary to a NY Times report in May 1994, watermelons are not known to "explode." Still, these are not watermelons you want to buy or eat.

The disease is caused by the bacterial pathogen Acidovorax avenae subsp. citrulli (Aac), formerly considered to belong to the genus Pseudomonas. The disease cycle starts with infected seed, the most important source of primary inoculum. Since watermelons are typically seeded in flats, with many seedlings close together, maintained under high humidity, and watered by overhead irrigation, the pathogen can spread very easily from one seedling to the next, and a single infected seedling can infect the entire tray. The infection of the fruit actually occurs very early on, after the flower opens. The symptoms don't show up until much later. The infected fruit cracks open, seeds come out, can germinate in the soil (volunteer seedlings), and become a source of secondary inoculum.

BFB was first reported in Georgia in 1965, 4 years later it was observed at a research farm in Leesburg, Fl. In 1978 a strain was described that caused seedling blight, and did not show a hypersensitive response (HR) on tobacco or tomato. In 1987 there was an outbreak in the Mariana Islands, and in 1989 a strain was recovered from commercial plantings of watermelons in Florida and Indiana. Contrary to the 1978 strain, this strain caused seedling blight and fruit rot, and elicited an HR on tobacco and tomato. Since 1992, many US states have reported BFB, and there has been a significant amount of yield loss. In addition, growers have filed lawsuits against seed companies, the seed companies have in return restricted watermelon seed sales. Seeds now come with a disclaimer; the growers needs to accept the risk of BFB, and agree not to file any lawsuits.

In 1995, management guidelines were established in Georgia to control the pathogen, and these seemed to limit the problem initially, but then in 1999 and 2000 there were substantial outbreaks. Either the guidelines didn't work, or there was something else going on. Because of the increased movement of seeds, BFB currently occurs worldwide.

In 1999 there were also outbreaks on different crops. BFB was no longer restricted to watermelon, but was observed on canteloupe, melon, and pumpkin (oh no! What does that mean for Halloween?). There were often no obvious symptoms, other than a few small spots on the rind. However, below those spots, the rind doesn't develop, and the fruit goes bad. Some other hosts that the pathogen has been found on include cucumber, honeydew, hami melon, squash, bitter and bottle gourd. Seed transmission has been confirmed for several of these.

Dr. Walcott's lab investigated the genetic diversity of Aac. Using several different techniques, he discovered that at least two groups can be distinguished. Group I strains show less genetic variation than group II strains. Groups I strains have similar, moderate severity on a number of hosts, while group II strains is much more severe on watermelon, while being moderately aggressive on other hosts. The genome of a group II strain has recently been sequences, and annotation of the genome is currently in progress. Dr. Walcott is investigating the possibility of sequencing a group I strain too, so that differences at the genomic level can be correlated with differences in virulence.

Currently, the production of watermelon seed is not really conducive for Aac spread. The seeds are produced under cool, dry conditions, the seed production fields are visually inspected, and the seeds are tested for presence of Aac. But Aac continues to be a problem. Dr. Walcott therefore studied the mechanisms of seed infection. He figured there were 3 possibilities:
1. The bacteria penetrate through the ovary wall. This is unlikely, symptoms would be apparent.
2. The bacteria infect the plant systemically via the vascular system. However, there is no evidence of systemic infection.
3. The bacteria penetrate through the flower parts. Hmmm. This is promising.

Dr. Walcott considered that the bacteria may land on the stigma, move through the style, and end up in the ovary, and thought it the most likely possibility.

He took symptomless fruit, harvested the seed, and found that Aac can associate with seeds, without any symptoms on the fruit. He developed transgenic Aac bacteria expressing green fluorescent protein (GFP), so that he could track the bacteria easily. And indeed, he found the bacteria present in seed tissue, and he could track the bacteria following the path described above. He also noted that this type of infection is not unique to Aac, there are other pathogens that can do this. Using the GFP-tagged bacteria he also found out that the bacteria take about a week to travel from the stigma to the ovary via the pistil pathway. Once the bacteria reached the ovary, however, replication of the bacterium stopped. This explains the lack of symptoms.

Dr. Walcott discovered that bacterial motility was not important for colonization of the seeds, but that pollination was necessary. He also investigated the role of pollinating insects, and found in one experiment that bees did seem capable of spreading the infection, but these results need to be verified with additional experiments. The problem with this theory in practice is, however, that in commercial seed production fields, most pollination does not occur by bees, but by hand, so the bees may not be sufficient to explain the outbreaks seen.

Towards the end of seminar, Dr. Walcott discussed some of the experiments he has done to investigate the possibility of biological control. Although he found a good level of control with at least one biological control agent, this is not good enough for watermelon seed production with a zero-tolerance of Aac.

Further reading

Fessehaie, A., and Walcott, R.R. (2005) Biological control to protect watermelon blossoms and seeds from infection by Acidovorax avenae subsp. citrulli. Phytopathology 95:413-419.

Gitaitis, R.D. and Walcott, R.R. (2007) The epidemiology and management of seedborne bacterial diseases. Ann. Rev. Phytopathology 45: 371-397.

Lessl, J. T., Fessehaie, A. and Walcott, R. R.. 2007. Colonization of female watermelon blossoms by Acidovorax avenae subsp. citrulli and the relationship between blossom inoculum dosage and seed infestation. J. Phytopathology 155:114-121.

Walcott, R.R. (2005) Bacterial fruit blotch of cucurbits. The Plant Health Instructor. DOI:1094/PHI-I-2005-1025-02

Late-season vine decline of melons

The seminar disclaimer applies to this post.

This week our departmental seminar was given by none other than the current President of the profession organization for plant pathologists, the American Phytopathological Society (APS), Dr. Raymond D. Martyn. Dr. Martyn is a homegrown graduate, who did his PhD research on biological control of waterhyacinths. He then moved to Texas A&M, where he was a professor for 20 years, and his research focused on soil-borne diseases of melon. He then moved to Purdue University as Head of the Botany and Plant Pathology Department.

Dr. Martyn is no longer involved in active research but this has given him plenty of time to digest the work he did over the years.

During the past 20-25 years a dramatic increase in late-season vine decline (LSVD) of melons has been observed. This involves all members of the cucurbit family of plants, for example watermelon, squash, and muskmelon. LSVD is a generic term used to describe a set of symptoms that include the sudden death of cucurbit foliage about 2-3 weeks before harvest. This period is critical for the accumulation of sugars during the final ripening stages of the melons, so the vine death results in major losses.

There is no single cause for LSVD, nothing that correlates exclusively with vine decline. Sometimes there are pathogens involved, other times not. Vine decline has reportedly been associated with presence of fungi, viruses, bacteria, application of herbicides, and more. The fungus Monosporascus cannonballus, is one of the major fungal pathogens found to be associated with LSVD.

M. cannonballus is a fungus that forms fruiting bodies called perithecia on the roots of the melons. These perithecia have structures in them called asci (singular: ascus). Fungi belonging to the ascomycota typically have asci with 8 ascospores, M. cannonballus is different in that each ascus only has one ascospore, but this spore contains 8 nuclei (it's a called a multinucleate ascospore). Sometimes 16, but mostly 8. Looking at the picture below, it's easy to see where the fungus gets its name from, the perithecium is almost perfectly round and smooth. The ascospores have very thick walls, and are hard to germinate: they are dormant spores. M. cannonballus was first reported in 1970 as a saprophyte (not a pathogen) on canteloupe, in 1983 it was first reported as a plant pathogen in Israel, and in 1989 it was first reported within the US by guess who... Dr. Ray Martyn.

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In the greenhouse, M. cannonballus can infect many different plant species. When a fungus can infect many species, it is said to have a "wide host range." However, M. cannonballus is only a problem on watermelon and muskmelon in the field, so for practical purposes M. cannonballus has a narrow host range. Dr. Martyn studied potential biocontrol methods. He looked at ways in which the disease can be managed using organisms. In this case, M. cannonballus changed in culture, and became hypovirulent, meaning they caused a lot less disease than the original fungus. He investigated whether he could decrease the damage caused in the field by adding the hypovirulent fungus, which would "mate" with the disease-causing fungus, resulting in hypovirulent offspring.

Dr. Martyn authored an article for the APS Education website on Monosporascus root rot and late-season vine decline of melons.

A number of other pathogens were studied as possible causes of late-season vine decline, but all turned out to be fairly weak pathogens, that only caused damage when the plant was very young. In LSVD, the plant is mature, and much stronger, and these pathogens were not responsible for the disease symptoms. In addition, Monosporascus is not the cause of LSVD everywhere in the world. It is reported only in areas that are fairly warm.

Since LSVD is a fairly recent syndrome, Dr. Martyn considered the changes in melon production in the past 25 years. These are:
1. Melon production switched from open-pollinated plots to the use of hybrid melon cultures.
2. Instead of direct seeding into the ground, current production techniques almost exclusively use transplants (seedlings are grown in small trays, and transferred into the ground when they are older).
3. Planting used to be in bare ground, and irrigation systems used furrows, currently plastic mulch and drip irrigation are used.
4. Plants are planted much closer together than in the past.

Dr. Martyn proposed during the seminar that these changes in melon culture practices result in compromised and restricted root systems on the plants. When melons are seeded directly into the ground, the initial root that is formed when a seed germinates, the tap root, continues to grow deep into the soil, as deep as 6-8 feet. A large number of secondary and tertiary roots form along the length of the tap root, allowing the plant to extract water and nutrients from deep in the soil. When seed is germinated in little seedling trays, the tap root grows to meet the bottom of the tray and starts growing in a circle. Once the plant is transplanted into soil, the taproot never recovers, and dies off. Secondary roots that are formed after transplanting do not grow deeply into the soil, but remain about 4" or so below the ground level, and grow essentially horizontally. This drastically limits the availability of water and nutrients to the root system, because the roots do not reach deep enough into the soil.

The compromised root systems have a hard time keeping up with the demand for water of the mature plant, especially during the latter stages, when fruits need to accumulate sugars. Minor pathogens and additional stress, for example lack of water, cause rapid death of the plant in the final stages of fruit development, when the plant needs the resources the most.

Dr. Martyn investigated using cone-tainers, which are very long containers to start seedlings, and they allow for much more growth of the tap root before transplanting, and are so long that the tap root does not reach the bottom before transplanting occurs. After the seedling is planted in soil, the tap root can continue to develop.
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In conclusion, LSVD correlates with cultural changes, resulting in vulnerable root systems that cannot handle multiple stresses.