Didymella bryoniae (anamorph: Phoma cucrubitacearum) is the causal agent of gummy stem blight (GSB), a disease affecting members of the family Cucurbitaceae. The cucurbit family includes economically important crops such as watermelon (Citrullus lantanus), muskmelon (Cucumis melo), cucumber (Cucumis sativa), and pumpkin (Cucurbita pepo and Cucurbita maxima). Infection can occur on the leaves, the stems, and the fruit. It is also suspected to infect the seed as well. When infection occurs on the fruit, the disease is often called black rot (Zitter, 1992).
Taxonomy and phyologeny
The original name, Sphaeria bryoniae was first described by Fuckel in 1870. Since then, there have been many additional classifications of this ascomycete (Index fungorum 2013). Didymella bryoniae was first observed as a pathogen in France in 1891 on a chinese variety of cucumber. The pathogen was reported in the US the same year in Delaware on watermelon, squash, muskmelon, and pumpkin (Chui, W.F. and Walker, J.C. 1949a). F.B. Chester described the fungus found on infected watermelon in Delaware. After isolating the culture and re-infecting new plants, he concluded it was in the genus Phyllostica and named it Phyllostica citrullina (Chester 1892). In 1949, W.F. Chui and J.C. Walker released two publications looking at the morphology and variability (1949a) along with a paper describing the physiology and pathogenicity of the pathogen collected from infected fruit (1949b). Chui and Walker classified the fungus as Mycosphaerella bryoniae. (1949a)
The imperfect fungal stage has been described as Phoma cucurbitacearum. This was first described as Sphaeria cucurbitacearum by Lundae in 1823. (Index Fungorum, 2013) Didymella bryoniae is in the phylum, Ascomycota Class, Dothideomycotes, Order, Pleosporomycetidae Family, Pleosporale. It should be noted that the latin term “Incertae sedis” is often included in the classification. This term suggests that the placement of the fungus in the classification is uncertain. (Index Fungorum, 2013).
General classification scheme:
Kingdom: Fungi
Phylum: Ascomycota
Class: Dothideomycetes
Subclass: pleospormycetidae
Order: Pleosporales
Family: Didymella
Genus: D. bryoniae
Species:
The genome of Didymella bryoniae has been sequenced in several regions including the Internal Transcribed Spacer (ITS) region to identify the species. Additionally, the species has been separated by a region for virulence, named the Phoma I and Phoma II groups. (Somai et al, 2002). There has also been some molecular characterization of the resistant isolates for polymorphisms of importance to fungicide resistance. (Arvenot et al, 2012).
Morphology
Didymella bryoniae (anamorph: Phoma cucrubitacearum) is a homothallic ascomycete. There are two stages of spore formation, sexual and asexual. Sexual spores are produced by structures called perithecia, which give rise to ascospores. Another form of ascocarp is possible, known as a pseudothecia, although this is rarely observed. Ascospores are believed to serve as the primary inoculum and are better suited to wind dispersal. Asexual spores, called conidia, are produced in structures called pycnidia. Often, both types of spores can be found in the same lesion. Conidia are typically single-celled which tend to be 3 times longer than they are wide. Conidia are also believed to be better adapted to shorter distance transmission, such as rainsplash. (Wehner, 2008) Most experts agree that the conidia serve as a secondary spread of the disease. (Paret, Dufault, and Olson, 2011).
In culture
In culture, hyphae may appear brown, white, gray or green. The culture will form concentric circles with the altering of day and night light conditions. Hyphae are septate and black pycnidia are often found in the culture. The genome of Didymella is known to mutate quickly. (Wehner, 2008). Spore morphology appears to depend on the structure that formed the aforementioned spores. Perithecia diameter can be as large as 200 µm with a narrower osteole. Ascospores arising from Perithecia tend to be 5-8 µm wide with an overall length ranging from 15 to 21 µm. Spores are monoseptate and hyaline in color. Pseudothecia diameter vary between 125 to 215 µm in diameter and appear dark and have only been observed on the stems of infected cucurbits. Ascospores arising from pseudothecia are smaller, with widths ranging from 4-6 µm and a length of 14-18 µm. Spores are monoseptate and hyaline in color. One distinctive characteristic of these ascospores is the top cell above the septum tends to be larger than the bottom. Both perithecia and pseudothecia produce spores in a sac or asci which contain eight genetically distinct spores (Chiu and Walker 1949a).
In the case of asexual spore formation (Phoma), the pycnidia also appear dark, with a smaller diameter of 120-180 µm. These fruiting bodies have been observed on the leaves, stems and on the fruit. Conidia also appear hyaline in color and range in length from 6 to 13 µm. Conidia may be monoseptate or nonseptate. (Paret, Dufault, and Olson, 2011).
In field (Signs and symptoms)
Symptoms of gummy stem blight can span a wide range, depending on the species of cucurbit. Leaf symptoms are the most common, which may be round, water-soaked lesions and sometimes are surrounded by a yellow halo. Necrotic scorching around the interveinal region is also possible. Stem symptomology includes water-soaked lesions, which may turn tan over tie. A classical symptom is the red or black beads of exudate found along the stem, hence the disease name (Zitter, 1992). This is caused by a pectolytic enzyme called polygalacturonase. This is an important virulence tool for the pathogen; this enzyme allows for the cell walls of the host to be degraded. (Chilosi, G and P Magro 2002). When the disease is present on the fruit, it is sometimes called black rot. This becomes economically important especially postharvest. As with leaf symptomology, the symptoms may differ depending on the species. Lesions on butternut squash (Cucurbita moschata) appear bronze and in irregular shapes. Lesions may also be raised or corklike. The characteristic black rot becomes evident during the storage process. Pycnidia may be present, which will appear as black specks on a ring pattern. This can appear on leaves, stems, or fruit. Perithecia and pycnidia can be found readily, sometimes within the same lesion. (Zitter, 1992).
Ecology
Many isolates of Didymella bryoniae are pathogenic to plants. (Wehner, 2008). This organism is often associated with the production of cucurbit crops, which include economically important vegetables such as cucumber, watermelon, squash, pumpkin, luffa, and muskmelon. (The plant list 2010). D. bryoniae gets its carbon by parasitizing the aforementioned crops as hosts.
In the United States, Didymella bryoniae tends to be found more often in the southern US compared to the northern counterpart. The major range-limiting factor is the overwintering period. This fungus has been reported in other countries, including major cucurbit producing countries. D. bryoniae is considered a facultative necrotroph. While there is no evidence that D. bryoniae can be saprophytic, crop debris can harbor this pathogen. Infected debris can be a source of inoculum from one year to the next. In many fields, infected vines may harbor D. bryoniae for up to 24 months (Keinath 2008). However, with good crop rotation, appropriate tillage and elimination of volunteer plants, this is a rare source of D. bryoniae. (Keinath 2013)
More often, D. bryoniae inoculum enters the field is through infected transplants. This entails a grower buying infected transplants and planting them into the field. Additionally, transplants are at a greater risk of infection since their cotyledons tend to be highly susceptible in watermelon (Sitterly et al 1996). Furthermore, seedborne infection has been observed in watermelon (Hopkins, 1996) and muskmelon (Sudisha ,Niranjana, Umesha, Prakash, and Shetty 2006). This could prove to be a major source of infection without proper seed treatment.
Moisture and temperature are important factors in infection, as is the case with many plant pathogens. These factors are also important in the biology of the pathogen and play a role germination, colonization, and sporulation of the conidia. Optimum infection temperatures range from 61-75o F with a high relative humidity (>85%). Symptoms tend to occur 7-12 days after spore germination. Additionally, wounding, either mechanical or biological, can lead to increased incidence of Gummy Stem Blight infections (Paret, Dufault, and Olson, 2011).
Relevance to humans
Commercial cucurbit production in the US was estimated at 109 million metric tons on nearly 230,000 hectares in 2007. The cucurbit production in 2007 was estimated at $1.43 billion. In fact, in 2004, Watermelon, muskmelon and cucumber are in the top 15 vegetables produced (total production), ranked at 4th, 11th, and 13th respectively (Cantliffe, Shaw, and Stoffella, 2007). With such a large production scale and a high value, it becomes clear that any threat to the industry should be taken seriously.
Growers have limited management options depending on the crop. Watermelon is the most susceptible crop. (Paret, Dufault, and Olson, 2011). While there is an array of resistant varieties available resistant to other pathogens on watermelon, such as anthracnose (Colletotrichum orbicularaea) and downy mildew (Pseudperonospora cubensis), there is no commercially available variety which has a resistance package to D. bryoniae. Breeders have identified some resistance factors present in some populations. There is an ongoing breeding trial at the University of Georgia addressing this problem (Langston and McGregor, 2010). As a result, D. bryoniae has emerged as a major pathogen in watermelon. (Sitterly et al, 1996).
It is believed that the pathogen can also be transmitted via infected seed (sitterly et al, 1996). Growers should always start with clean seed. Most commercial seed companies will test each lot for contamination. Wet seed treatments, such as fermentation, peroxyacetic acid, and hydrochloric acid washes were found to significantly reduce incidence of both Acidivorax (bacterial fruit blotch) and gummy stem blight (GSB) (Hopkins 1996).
Without a resistant variety available for growers, the main tool in their disease management is chemical control through the use of fungicides. One major challenge with all fungicides is the pathogen development of resistance. This particular pathogen has been through several cycles of resistance. The first recorded resistant isolate of Didymella was found in Greece in 1981 and subsequently confirmed to be in the US in 1995. These strains were equally virulent to their sensitive counterparts. (Keinath and Zitter, 1998). There is widespread resistance to modern fungicides among many classes of chemistries. There is resistance recorded in three of the four major fungicide classes labeled for control. (Keinath, 2012).
To conclude, the management of this disease remains to be a challenge. Growers are limited to fungicide application, which is costly and becomes ineffective over time. Further research is needed on the fungicide resistance and how to best manage resistant strains. Additionally, cultural practices are important to implement to reduce inoculum loads from season to season. Commercial varieties with resistance to D. bryoniae would also reduce disease severity for growers.
Didymella bryoniae, syn. Mycosphaerella melonis, is an ascomycete fungal plant pathogen that causes gummy stem blight on the family Cucurbitaceae (the family of gourds and melons), which includes cantaloupe, cucumber, muskmelon and watermelon plants.[1][2][3][4] The anamorph/asexual stage for this fungus is called Phoma cucurbitacearum.[2] When this pathogen infects the fruit of cucurbits it is called black rot.[2]
The first symptoms appear as grayish green, circular spots between the veins of the leaf lobes.[1] With age these spots darken to brown and black.[1][2] Lesions begin to develop on vines at the vine nodes and then elongate into water-soaked streaks, and these streaks are pale brown at first but turn gray with time.[1] The petioles and stems eventually become necrotic and often shrivel. Eventually all infected vines will become necrotic and occasionally the plant dies due to wilting and defoliation.[1][5] Another common sign following the stem lesions is a red to amber colored ooze.[6] Some regions report the presence of small pseudothecia as black specks inside the cankers.[7]
Gummy stem blight can be confused with anthracnose, which is caused by a fungal plant pathogen called Colletotrichum lagenarium.[1] To distinguish between anthracnose and gummy stem blight, gummy stem blight leaf lesions are darker, target-like and less deteriorated than anthracnose lesions.[1] Newly infected plants will begin to show symptoms within 7–12 days.
In vitro, the fungal growth on an agar plate looks rough and undulated.[5] When grown in vitro on agar, the fungus produces a white to olive-colored mycelium. In latter periods of growth, the mycelium is an olive to dark green or black color.[5]
Didymella bryoniae survives on deceased vines, crop debris and on seeds in between seasons and D. bryoniae can survive for 5 months on the soil surface in winter.[2][4] The fungus develops best under moist conditions, and cotyledons and young watermelon/melon leaves are especially susceptible to the fungus.[2] D. bryoniae produces ascospores (meiotic spores) in perithecia and conidia (mitotic spores) in pycnidia and both of these spores are dispersed by rain/rain-splash and UV light is needed in order for the fungus to sporulate.[3] Ideal ascospore dispersal occurs after nightly rainfall and dew periods.[2] In order to infect, ascospores must land on leaves that have free-standing water on them.[2] Next the ascospores penetrate through the leaf cuticle.[2] Stems may be infected by D. bryoniae ascospores through stem wounds or by the extension of leaf lesions.[2] Fruits are penetrated through wounds and pollination flower scars.[2] Conidia are produced on the lesion sites of leaves and stems. Certain Cucurbita species are resistant to D. bryoniae but become vulnerable once they mature.[2]
Didymella bryoniae is common in the Southern U.S. and other subtropical or tropical locations.[2] Most infections occur during rainy/wet seasons, in which the humid is greater than 90% and the temperature is roughly 20–24 °C.[8] Humidity seems to be a larger factor than temperature when it comes to infection success.[2] D. bryoniae can also be found in temperate regions, especially where winter squash and pumpkins are grown.[2] This pathogen is also common in greenhouses where cucumbers are grown.[2]
D. bryoniae can be spread by the transfer of conidia through a variety of fashions. The most common forms of transfer for these conidia are through the air and water splashing. The fungus is capable of surviving in dead plant tissue giving it the ability to infect the following crop planting.[9] The pathogen requires an entry site on the plant in order to infect so areas that also experience issues with pests are at higher risk.
In vitro, D. bryoniae does not form pycnidia without UV-light but if cultured in the presence of UV light and darkness, conidia/pycnidiospores produce mycelium rapidly.[10]
The standard management practice for D. bryoniae is to use pesticide treated/pathogen-free seeds and to rotate crops on a 2-year cycle to reduce inoculum prevalence.[2] There are no commercially acceptable resistant cucumbers, melons or watermelons available yet on the market, but some plant breeders have identified D. bryoniae resistant genes, such as the gene db in watermelon.[2][11] Regular benzimidazole fungicide applications can control this pathogen, but certain D. bryoniae isolates have been found to be resistant to benzimidazole fungicides in greenhouse settings and in the field.[2]
Along with fungicides, it is important to have proper ventilation and irrigation practices in greenhouse settings.[2] Proper irrigation and ventilation can be utilized to prevent water buildup on leaves.[2] Also to prevent disease onset in greenhouse settings, use UV-absorbing vinyl film, to prevent fungal sporulation.[12]
Currently cultural practices and fungicides work well in greenhouses and in the field only if D. bryoniae is diagnosed in the early stages of disease development.[4] Molecular tools such as polymerase chain reaction (PCR), PCR-enzyme-linked immunosorbent assay and magnetic-capture hybridization multiplex real-time PCR are used to diagnose D. bryoniae in the early stages disease development, although these molecular tools may only be useful for specific isolates of D. bryoniae.[4][13][14]
The United States consumed 15.69 pounds of watermelon per capita in the year 2018 after a rise in both total imports and locally produced watermelons.[15] Florida and Georgia characterized 35 isolates of Didymella and phoma spp. Associated with symptoms of gummy stem blight on watermelon. These two states produced 42% of the United States total watermelon value in 2013, and a combined 20,000 hectares in total farm area. Florida alone produced 907 million pounds of watermelon in 2019[16] meaning that this pathogen could have a direct effect on at least 25% of domestic watermelon crop in the United States.
Didymella bryoniae, syn. Mycosphaerella melonis, is an ascomycete fungal plant pathogen that causes gummy stem blight on the family Cucurbitaceae (the family of gourds and melons), which includes cantaloupe, cucumber, muskmelon and watermelon plants. The anamorph/asexual stage for this fungus is called Phoma cucurbitacearum. When this pathogen infects the fruit of cucurbits it is called black rot.
