Diplodia seriata
(grapevine trunk disease)

Diplodia seriata is a cosmopolitan and plurivorous fungal species occurring on woody hosts belonging to many plant genera and families (Punithalingam and Waller, 1973; Phillips et al., 2007; Slippers et al., 2007). The fungus is encountered in many habitats, but has a primarily temperate distribution and is present on most continents.

D. seriata causes canker, dieback, fruit rot and leaf spot diseases on economically important forest and horticultural species (Farr and Rossman, 2020). Reports of the virulence of this pathogen vary depending upon the crop, varieties and hosts involved and it is often regarded as a stress-related pathogen taking advantage of weak or stressed plants. In common with other members of the Botryosphaeriaceae, D. seriata is capable of living endophytically inside plants (Crous et al., 2006; Slippers and Wingfield, 2007) and latent infections of fruits can result in storage rots. The pathogen is dispersed through both pycnidia and ascospores with conidia regarded as the most important inoculum source for short-distance spread. Infection is through wounds, natural openings, or direct penetration of the host tissue. There is no evidence that this species is seedborne although some members of the Botryosphaeriaceae have been shown to be present in seeds (Gure et al., 2005). The extensive host range of this species means that it is more likely to become established in new areas, as establishment will not depend on the presence of specific hosts. The widespread distribution of this species is presumably as a result of the word-wide movement agricultural, forestry and ornamental plants.

Currently no regional plant protection organization consider D. seriata to be a quarantine pest, possibly due to its already extensive distribution. As with most canker fungi these organisms can survive in a reproductive state on woody host material. The prolonged latent infection, or endophytic phase on some hosts means that the fungus can pass undetected by quarantine systems in traded living plants, fruits, and other plant parts, illustrating the phytosanitary shortcomings related to the detection of endophytic plant pathogens. The colonization potential for D. seriata is high because of the wide host range and diversity of environments it could encounter upon entry. The pathogen is damaging to a wide variety of hosts especially if new more virulent strains are introduced that affect ornamental or high value plantings.

The fungus D. seriata, known for many years as Botryosphaeria obtusa, is an important pathogen of apples causing frog-eye spot, black rot, canker and shoot dieback. In addition to apples, B. obtusa has been isolated from at least 34 different hosts (Punithalingam and Waller, 1973). In recent years it has been recognized as a pathogen of Vitis vinifera in Europe (Phillips, 1998, 2002; Urbez-Torres, 2011), Australia (Castillo-Pando et al., 2001) and South Africa (van Niekerk et al., 2004).

Punithalingam and Waller (1973) reported this fungus to be isolated from 35 different plant genera, but the current number of known hosts is much greater. According to the Systematic Mycology and Microbiology Laboratory Fungal Database (Agricultural Research Service, United States Department of Agriculture) there are 209 named species representing 151 genera of herbaceous and woody hosts associated with this pathogen including synonyms (Farr and Rossman, 2020). The Herb IMI database (Herb IMI Database, Royal Botanic Gardens, Kew, UK) lists 47 species and 40 genera as being associated with Peyronellaea obtuse (an erroneous synonym for D. seriata). Combining the herbarium lists results in 241 named species from 163 genera that have been reported associated with this fungus. There are however some question marks over the authenticity of some of these associations given the complex taxonomy of this species and the unreliability of morphological characteristics for identification. Moreover, in some instances the fungus may have been growing saprophytically on dead material rather than acting as a primary pathogen.

Genetics

The genetic diversity of a species is highly influenced by the relative contribution of its asexual and sexual reproduction forms. Reproduction of Botryosphaeriaceae species is believed to be mainly by asexual means (Phillips, 2002; Úrbez‐Torres, 2011) and sexual fruiting bodies are rarely found in the field (van Niekerk et al., 2004). Nevertheless, it has been reported that compatible genotypes can exchange genetic material through parasexual recombination (Leslie, 1993). The genetic diversity of D. seriata populations on grapevines in Spain was investigated using the inter‐simple sequence repeat (ISSR) technique (Elena et al., 2015). This study found that isolates from different geographic origins or from different hosts were not grouped in genetically distinct clusters. This suggests that the isolates are genetically similar regardless of their geographic and host origin. A similar conclusion was reported by Phillips et al. (2007), who found no correlation between the host origin of different D. seriata isolates and the clustering structure obtained from a phylogenetic study of this species based on ITS sequence data. The pathogenicity and virulence of D. seriata is usually evaluated through its ability to cause brown necrotic lesions in the wood and the length of these lesions, respectively, there are conflicting reports as to the virulence of D. seriata on hosts form different geographic locations. These differences may be due to variations in virulence between strains, or they may be a result of the incomplete knowledge of the taxonomy of the genus, which in turn hampers accurate species recognition and identification.

Life-cycle

The fungus overwinters in fruiting bodies (pycnidia and perithecia) on dead bark, dead twigs or mummified fruit. It has also been demonstrated to survive endophytically inside some hosts, where it can invade almost any dead, woody tissues.

In the spring, pycnidia and perithecia are trigged to release conidia and ascospores, under high humidity and during wet periods throughout the growing season. The spores are dispersed by splashing rains, wind and insects. The pathogen invades the tissue primarily through wounds, although in some hosts entry through natural openings such as lenticels and stomata is possible as well as direct penetration. Depending upon the host, the conidia can infect a variety of organs including leaves, the calyxes of blossoms, tiny fruit, and wounds in twigs and limbs. Infections of fruit and wood may not become visible for several weeks. The spores germinate at temperatures between 15 and 37°C and grow between 5 and 37°C. Infection is favoured by conditions that can stress the plant such as drought, frost damage, hail damage, poor nutrition and poor pruning practices. 

Many of the host plants attacked by D. seriata are infected through wounds, therefore much of the research into natural enemies has concentrated on wound protection products. The majority of the commercial products available make use of species of Trichoderma which are antagonistic to D. seriata. Despite extensive research and increased availability, there has been limited adoption of biocontrol agents in commercial agriculture, mainly due to inconsistent and unpredictable performance. For grapevine pruning wounds the physiological state (dormant or active) of the vines at pruning can affect how the Trichoderma spp. colonises the wound. Delayed pruning may result in excessive sap bleeding which may dislodge any wound protectants applied immediately after pruning.

There are no references in the literature of D. seriata being seed transmitted, however this species, in common with other members of the Botryosphaeriaceae, can live endophytically inside plants and latent infection of fruits is commonly reported for D. seriata. Despite there being no records of seed transmission of D. seriata, there is evidence that at least some members of the Botryosphaeriaceae family can be transmitted in seed (Gure et al., 2005), however, there is little evidence that these seed infections result in systemic infections in the plants as they develop (Slippers and Wingfield, 2007).

D. seriata can be identified using classic and molecular biology methods. In classic identification, the fungus is first isolated into pure culture using aseptic techniques and grown on standard agar, such as half strength Potato Dextrose Agar (PDA). Colony growth, colour, conidiophore, and conidial morphology are used to distinguish species (Crous et al., 2006; Urbez-Torres et al., 2006; Pitt et al., 2010). Classic identification of D. seriata relies on morphological features, however, recent taxonomic evaluations by several researchers have shown that these characteristics are variable and often overlap between species (Phillips et al., 2013).

The accurate identification of Botryosphaeriaceae is therefore best achieved by DNA sequence data rather than relying on morphological descriptions. Phillips et al. (2013) recommended that at least two loci, the internal transcribed spacer (ITS) region, and the translation elongation factor 1-alpha (tef1α), be used for species separation. However, Slippers et al. (2013) recommended the use of four loci, including the ITS region, tef1α, beta-tubulin (tub), and the RNA polymerase II (rpb2), as these loci provide better resolution to distinguish cryptic species. Unfortunately, the amplification of rpb2 is challenging and subsequently there is lack of data for comparisons (Slippers et al., 2013). Procedures and protocols for DNA isolation and sequencing are explained in detail by Alves et al. (2004).

D. seriata is difficult to diagnose definitively based on symptomology alone as the symptoms it produces vary depending upon the host and environmental conditions and are often not unique. Moreover, D. seriata may be present in apparently healthy-looking plants as a latent infection, or colonise dead woody parts damaged by other fungal pathogens, insects or abiotic agents. Where present, the fruiting bodies of the fungus on mummified fruit, cankers or dead wood are a reasonable indication of its presence, however other closely related fungi can produce similar structures so this is not conclusive unless backed up by microscopic analysis, or better still, molecular testing.

This pathogen can induce cankers, diebacks, and fruit rots on a wide variety of hosts and these symptoms can easily be confused with those caused by many different fungal pathogens and abiotic disorders. Given the enormous host range of this pathogen, it is not within the scope of this document to attempt to describe all the possible organisms with which it could be confused. Therefore, two of the most important economic hosts, grapes and apples have been selected for further discussion.

D. seriata has been implicated along with several other fungi in the condition known as ‘grape vine trunk disease’ (GTD). Affected vines show external symptoms that include a general and progressive decline (delayed budburst, dead buds, dieback, cankers, stunted development, chlorosis, apoplexy) as well as internal symptoms of brown streaking of vascular tissues and sectorial, or central necrosis. Based on the predominant organism responsible, GTD currently includes black foot, Esca, Eutypa dieback, Phomopsis dieback, Petri disease and Botryosphaeria dieback (Pascoe, 1998; Mugnai et al., 1999; Úrbez-Torres et al., 2006; Úrbez-Torres and Gubler, 2011; Lecomte et al., 2012; Mondello et al., 2018).

D. seriata is one of the most cited Botryosphaeriaceae species occurring on grapevines worldwide and is frequently associated with the ‘black dead arm’ disease of grapevine (Larignon et al., 2001; Úrbez‐Torres, 2011). However, it is just one of at least 20 different species in the Botryosphaeriaceae occurring in grapevines (Úrbez-Torres, 2011), the most common other species being Neofusicoccum parvum, Lasiodiplodia theobromae and D. mutila [Botryosphaeria stevensii]. Therefore, what is commonly referred to as Botryosphaeria dieback of grapes is a complex of different fungi that cannot be distinguished in the field based on symptomology. Moreover, symptoms such as dieback, dead spurs, stunted shoots, and bud mortality are shared with multiple trunk diseases that often occur in mixed infection within the vineyard and even within an individual vine. It is sometimes possible to distinguish Eutypa dieback from D. seriata dieback by the presence of foliar symptoms, but these are not always present.

Differentiation of the different members of the Botryosphaeriaceae involved in GTD is possible using isolation and morphological examination using the light microscope, but this is complicated by the overlapping characteristics of these pathogens and molecular-based techniques are therefore the preferred method for accurately ascribing species.

D. seriata is also an important disease of pome fruits, where it attacks the leaves, stems and fruits. Leaf lesions are referred to as frogeye spot due to the characteristic dark-brown concentric rings surrounded by a purple margin that develop around a light brown-to-grey centre, giving it a ‘frogeye’ appearance. These spots start off as small, purple specks that enlarge to form spots 3 to 6 mm in diameter. Black pycnidia, may develop on the upper surface in the centres of the older leaf spots which help to distinguish frogeye leaf spots from similar spots caused by spray injury. Stem symptoms of D. seriata begin as small, slightly sunken, reddish-brown areas that develop in the bark. These areas slowly enlarge and darken to form cankers with depressed centres and slightly raised and lobed margins. Cankers may also appear as a superficial roughening of the bark; or the bark may be killed and conspicuously cracked, especially at the margins. There are many potential causes of cankers in pome fruits, including other fungi, bacteria and mechanical injury, the development of black, pimple-like pycnidia and perithecia in older cankers is an indication that the canker may be caused by D. seriata, however cankers caused by another common apple pathogen Botryosphaeria dothidia cannot be told apart.

D. seriata also produces a fruit rot known as black rot of apples and pears. Initially the fungus infects the fruit through wounds caused by insects, hail or growth cracks, particularly at the calyx end of the fruit. At first, a light brown spot forms on the fruit which enlarges and is surrounded by a concentric zonation of lighter and darker brown colours. The rotted fruit finally turns black. The fruit symptoms are difficult to tell apart from rots caused by Colletotrichum gloeosporioides [Glomerella cingualata] and C. acutatum (bitter rot), however the development of ‘pimple-like’ fruiting bodies (pycnidia) on the surface of rotted fruit can help to distinguish between these pathogens. The fungus B. dothidia also causes a rot of apples that is known by the common name ‘white rot’, to distinguish it from the black rot caused by D. seriata. In practice these two diseases on fruits can be very difficult to tell apart. However, with black rot of apple, the flesh in the decayed portion of the fruit remains firm and somewhat leathery and the surface of the spot is not sunken. Conversely, the decay caused by the white rot pathogen is soft and forms a slightly sunken lesion.

Phillips et al. (2012) used molecular techniques to examine in detail the Botryosphaeriaceae attacking apples. This study revealed that D. seriata is a complex of species, two of which are associated with fruit rot and canker of apples and other Rosaceae, namely D. seriata and a species that they named D. intermedia. Both species are virtually indistinguishable based on morphology, which raises questions over previous reports regarding the black rot pathogen of apple (Phillips et al., 2012).

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.

Cultural and chemical management options for control of Botryosphaeria diseases are similar in many cropping systems including apple, blueberry, grape, peach and pistachio. Benzimidazoles, quinone outside inhibitors (QoI), and sterol biosynthesis inhibitors (DMI) are extensively used to treat the external symptoms of Botryosphaeria blight in apple (Brown and Britton, 1986), grape (Bester et al., 2007) and pistachio cropping systems (Ma et al., 2001; Ma et al., 2002). These products are applied either prophylactically, or as treatments applied to pruning wounds, as these serve as important entry points for infection. In addition to the synthetic chemical pruning treatments there are also several commercially available biological and botanical wound treatments. The biological products have already been outlined in the section ‘Notes on natural enemies’ and mostly make use of Trichoderma fungal antagonists that are painted on to the wounds. In addition to these several botanical products have been tested for their ability to manage D. seriata infections in grapevines, these products include chitosan oligosaccharide, garlic extract and vanillin. In field experiments all three were able to significantly reduce infection in pruning wounds by D. seriata and P. chlamydospora, with the most effective treatment being a mix of all three (Cobos et al., 2015).

Cultural control mostly relies on sanitation by reducing inoculum sources such as cankers, blighted shoots, mummified fruit, and pruning. In Californian vineyards delayed pruning is recommended as the current timing coincides with the highest periods of spore dispersal by fungi in the Botryosphaeriaceae.

Host resistance

Work is continuing to determine grape varieties with enhanced resistance to D. seriata and other members of the Botryosphaeriaceae. A study conducted by Guan et al. (2016) into the of genetic resistance of Vitaceae found differential susceptibility to wood necrosis caused by Neofusicoccum parvum and D. seriata. Several accessions of V. vinifera subsp. sylvestris, the ancestor of V. vinifera, were found to be more resistant to artificial inoculation than cultivars such as Chardonnay and Gewürztraminer. These findings suggest that creating new grapevine varieties with enhanced resistance to trunk pathogens is a realistic possibility.

Similarly, the host resistance of apples to black rot has been investigated by several authors experimentally and in the field. Biggs et al. (2004) tested 23 apple varieties for resistance and was able to classify the cultivars into three relative susceptibility groups - most susceptible: ʻOrinʼ, ʻPristineʼ and Sunriseʼ; moderately  susceptible: ʻSun-crispʼ, ʻGinger Goldʼ, ʻSenshuʼ, ʻHoneycrispʼ, ʻPioneerMacʼ, ʻFortuneʼ, ʻNY 75414ʼ, ʻArletʼ, ʻGolden  Supremeʼ, ʻShizukaʼ, ʻCameoʼ, ʻSansaʼ and ʻYatakaʼ; and least susceptible: ʻCrestonʼ, ʻGolden  Deliciousʼ, ʻEnterpriseʼ, ʻGala  Supremeʼ, ʻBraeburnʼ, ʻGoldRushʼ and ʻFujiʼ. 

For further information on the management of grapevine trunk disease, see Gramaje et al. (2018) and Mondello et al. (2018, 2019).

20/05/20 Original text:

Robert Reeder, CABI E-UK, Bakeham Lane, Egham, Surrey,  TW20 9TY, UK