Characterization of the Black Rot Fungus and an Update on Bacterial Wilt

Disease concerns for cucurbit growers, especially of pumpkins, run the gamut from early season concern for bacterial wilt, mid-season occurrence of powdery mildew and late season occurrence of gummy stem blight/black rot and Phytophthora blight. This discussion will cover the fungal disease commonly referred to as black rot and bacterial wilt, which until recently was only a concern for cucumber and melon production.

I. Gummy Stem Blight/Black Rot of Cucurbits - Introduction.

The soil- and seedborne fungus Didyinella bryoniae (the sexual stage) and its asexual stage, Phoma cucurbitacearum, cause gummy stem blight of cucurbits. This disease is known as gummy stem blight (GSB) when it attacks the foliage and as black rot when it affects fruits. The pathogen has worldwide distribution. The first issue that complicates identification of the causal agent from plant samples, including seed, is the fact that Phoma species other than P. cucurbitacearum have been associated with symptoms of GSB. Other Phoma spp. including Phoma exigua, however, are reportedly nonpathogenic or only mildly virulent to cucurbits, but may be pathogens of other crops such as potato. Another question that needed addressing was whether isolates that originated from a particular cucurbit species were more virulent on their hosts of origin, and whether there was any differential susceptibility among cucurbits (called specialization). Traditional methods to distinguish between virulent D. bryoniae and avirulent Phoma spp. include morphological observation (microscopic identification of the sexual structures, pseudothecia and ascospores, in the case of Didymella, and pycnidia and conidial spores in the case of Phoma spp.) and pathogenicity testing on various cucurbit species, where utilized in our studies. In addition, we used PCR (polymerase chain reaction) amplification with 3 specific primer sets (provided by colleagues in South Carolina) that allowed us to differentiate between D. bryoniae and Phoma spp. As a result, we separated the isolates into true Didymella bryoniae, D. bryoniae variant, which represents 90% or more genomic homology with type D. bryoniae, and Phoma spp. (which show less than 5% homology with either of the D. bryoniae types).

A. Lack of Specialization in the Gummy Stem Blight/Black Rot Organism.

Isolates of D. bryoniae and Phoma spp. were collected from four different cucurbit species: slicing cucumber (Cucumis sativus), muskmelon (Cucumis melo), jack­o’-lantem pumpkin (Cucurbita pepo), and butternut winter squash (Cucurbita moschata). Twenty representative isolates of either D. bryoniae or Phoma spp. were selected from across New York state (eastern, central, and western counties) and from different infected tissues (leaves, stems, and fruits) to increase the chance for diversity. Marketmore 76 cucumber, Topmark melon, Spirit pumpkin, Waltham butternut winter squash, and Sugar Baby watermelon seedlings were inoculated with the 20 isolates to determine if differences existed in pathogenicity and virulence among the fungal isolates. The tests showed that there was no indication of any preferential host specialization for the gummy stem blight/black rot organism, with similar infection levels noted on all tested cucurbits.

B. Genetic Variability of Didymella bryoniae and Phoma spp. Determined by Pathogenicity and PCR Primers.

Twenty-nine isolates of D. bryoniae and Phoma spp. were inoculated onto the susceptible muskmelon Topmark. Disease severity on individual plants was recorded 7 days after inoculation using a scale of 1 to 5 (1, no disease; 2, 1 - 25%; 3, 26 - 50%; 4, 51- 75%; and 5, 76 - 100% leaf area diseased, respectively). Twenty-three of the isolates were identified by D. bryoniae since they produced both ascospores and conidia. The remaining 6 isolates were identified as Phoma spp. and they produced only conidia. When inoculated unto Topmark melon, the isolates separated into three groups: D. bryoniae with high pathogenicity scores (approaching the maximum of 5); D. bryoniae with intermediate pathogenicity (now called D. bryoniae-variant with pathogenicity scores often in the middle 3.5 range); and Phoma spp. with low or nonpathogenic reactions (scores of 1.3 to 2.0) (Table 1). Pathogenicity of butternut fruit rind with the three organisms also behaved similarly. The PCR primers clearly separated the isolates into three groups similar to the pathogenicity results. Thus, recovery of the asexual stage of Phoma (pycnidia) from infected fruit, stems and leaves of cucurbits could be any of three possible organisms. The occurrence of D. bryoniae and D. bryoniae-variant appears to be unique to New York, but may occur in other northern locations.

II. Re-emergence of Bacterial Wilt as a Problem for Cucurbit Production.

Bacterial wilt, caused by the Erwinia tracheiphila, has long been known to be a factor for cucumber and muskmelon production. In fact, the threshold for cucumber beetles (the vectors) on cucumbers and muskmelons (because they are especially susceptible to wilt) is considered in some states to be 1 per plant (or less). Recently, summer squash and especially pumpkin plants expressing unusual symptoms, not indicative initially of a wilt disease, have become more common in New York. During the 1998 and 1999 seasons, bacterial wilt losses in pumpkin in New York have increased significantly throughout the state, and have approached 100% infection in some fields. Although such heavy losses are not common, the resurgence of this disease requires an update of factors responsible for disease development. We will examine host range, symptom appearance and timing of infection, vectors and the infection process, epidemiology, and overwintering means and importance of weed reservoirs.

At least four different symptoms appear on summer squash and pumpkin, and not all of these symptoms may appear together on any one plant. Plants may be infected over a period of time, which also adds to the range of symptoms expressed. Drought stress in 1999 also confounded field identification, but symptoms from the lack of moisture are different. The “typical” wilt symptoms on cucumber and melon consisting of flaccid (limp) leaves and then death of one or more vines, but this is not seen in pumpkins or summer squash. Instead, the signature for bacterial wilt on pumpkins consists of the following, with the monthly time of occurrence given in parentheses:

1. Distinct interveinal chlorosis (yellow) with the main leaf veins remaining dark green in color and the interveinal tissue eventually becoming necrotic or brown in color (dead) (June to September);
2. Shorten, tufted leaf growth than turns pale yellow to almost white in color with necrosis on the leaf margins (June, July). These symptoms mimic herbicide damage or even expression of Phytoplasma-like symptoms. This stunted growth habit may be the main signature feature of the entire plant when infected at the seedling stage or may appear at individual nodes of the infected vines when older plants survive and further plant growth occurs (August, September);
3. Collapse of one of more vines on a plant, but with symptoms 1 and 2 above. Such plants may support one or occasionally two fruits, but they fail to size and color properly, and are unmarketable (August, September);
4. Plants become necrotic, rot and die because of early plant infection, causing gaps in the planting, and a question in September as to what killed the plant s (usually begins in June and continues until September). The recent development of PCR (polymerase chain reaction) primers, designed to amplify the 16S rDNA (ribosomal DNA) of the bacterium, should aid in the detection of E. tracheiphila in plants and insects, and help to answer key epidemiological questions.

Thomas A. Zitter and Jessica L. Drennan, Department of Plant Pathology, Cornell University, Ithaca, New York 14853
Originally published: Proceedings. 1999. New England Vegetable & Berry Growers Conference and Trade Show. Sturbridge, MA.

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