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.

The genus Botryosphaeria was recently re-evaluated through a study of partial sequences of the LSU gene (Crous et al., 2006). This study determined that Botryosphaeria s. lat was composed of 10 phylogenetic lineages that represent individual genera. To avoid the introduction of new generic names, these authors chose to use existing asexual generic names for most of the lineages, and restricted the use of Botryosphaeria to B. dothidea (Moug. Fr.) Ces. & De Not. and B. corticis (Demaree & M.S. Wilcox) Arx & Müll. Consequently, the name Botryosphaeria is no longer acceptable for most of the species with Fusicoccum-like and Diplodia-like anamorphs including B. obtusa which has been named by its anamorph.

The anamorph of B. obtusa belongs in Diplodia Fr. because of its brown, aseptate conidia formed on phialides that line the inner wall of the pycnidial conidiomata and multiply via periclinal thickening, or annellations. However, the species differentiation within Diplodia has proven rather more difficult largely due to the lack of distinctive morphological features. This has resulted in Diplodia species being defined on the basis of host association and consequently a proliferation of species names. Unfortunately, the host is not a reliable means of species differentiation in the Botryosphaeriaceae and thus many of the names in Diplodia are likely to be synonyms (Slippers et al., 2004a).

As with the teleomorph the correct anamorph name for B. obtusa has also been the subject of much argument and confusion. In the past the debate has mainly revolved around the names Sphaeropsis malorum (Berk.) Berk. and S. malorum Peck. (Phillips et al., 2007). The anamorph S. malorum Peck. was introduced by Saccardo (1884) when he transferred S. malorum (Berk.) Berk to the genus Phoma, on the basis of its hyaline conidia. However, when Saccardo examined the S. malorum samples collected by Peck, he found them to be different from the Berkely collection, due to the production of brown conidia, and chose the name S. malorum Peck to represent them. Unfortunately, this name is an illegitimate, later homonym of S. malorum (Berk.) Berk. (1860) and is not recognized. The fungus S. malorum (Berk.) is itself a synonym of D. mutila, therefore neither of these names can be used for the anamorph of “Botryosphaeria” obtusa.  

The taxonomic position of the anamorph was recently clarified in a phylogenetic study of “B”. obtusa-type specimens conducted by Phillips et al. (2007). This study determined that D. seriata De Not. was the oldest name available for the asexual morph of what had been previously referred to as “B.” obtusa and this is the currently accepted species name. The circumscription of this fungus is however, further complicated by the likely existence of cryptic species (Phillips et al., 2012).

The genome sequences of two D. seriata isolates are available (Morales-Cruz et al., 2015 ; Robert-Siegwald et al., 2017). They are highly similar and have a typical Ascomycota genome organisation.

Infection by D. seriata is thought to occur through wounds, however, it is not clear whether these wounds are simple entry points, or whether they provide chemical signals that enhance spore germination processes. In addition, pathogenicity studies performed on woody hosts such as apple, peach and pistachio have demonstrated that these pathogens can also infect through natural openings such as stomata and lenticels or even penetrate host tissue directly (Michailides, 1991; Pusey, 1989; Kim et al., 1999). Both conidia and ascospores are infective, although ascospores are rarely found in the natural environment. The release of conidia is triggered by rainfall or humidity levels of 70% or above. Dispersal by rain splash is then thought to be over relatively short distances (Úrbez‐Torres et al., 2010, Baskarathevan et al., 2013). Phillips et al. (2007) selected a specimen on Vitis vinifera collected in Portugal (CBS-H 19809) as the epitype when proposing the anamorph and preferred scientific name for this pathogen. He gave the following description of the morphological characteristics. The conidiomata are pycnidial, separate or aggregated and confluent, immersed in the host, partially emergent at maturity, dark brown to black, ostiolate, non papillate, thick-walled, outer layers composed of dark-brown textura angularis, inner layers of thin-walled hyaline textura angularis. Conidiogenous cells 3- 5.5 × 7-10(-15) µm, hyaline, thin-walled, smooth, cylindrical, swollen at the base, discrete, producing a single conidium at the tip, indeterminate, proliferating internally giving rise to periclinal thickenings or proliferating percurrently forming 2-3 annelations. Conidia (21.5-) 22-27(-28) × (11-)11.5- 14.5(-15.5) µm, 95% confidence limits = 24.3-25.4 × 12-6-13.2 µm ( x ± S.D. of 50 = 24.9 ± 1.9 × 12.9 ± 1.1 µm, L/W = 1.9 ± 0.1) initially hyaline, becoming dark brown, moderately thick-walled (ca. 0.5 µm thick), wall externally smooth, roughened on the inner surface, aseptate, ovoid, widest in the middle, apex obtuse, base truncate or rounded.

D. seriata has been recorded worldwide on many different hosts of agriculture, forestry, or horticultural importance. The fungus was first described from Italy by Schweinitz (1832) as Sphaeria obtusa, and was found on dead stems of Jasminum. However, many past records relating to this pathogen are of questionable validity because of the previously confusing taxonomy and the unreliable nature of the morphological characters used in species identification. It is likely that much of the spread around the world was via the global trade of plants and plant products, but it is not possible to trace the historical routes of introduction.

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.

D. seriata has been associated with diseases such as fruit rot, dieback and cankers on a wide range of economically and environmentally important plants. There are too many hosts to discuss them all, so only a couple of economically important hosts are provided below, although it is likely that the symptoms of cankers and die back will be similar across many of the reported hosts.

On apple, the fungus affects a variety of plant parts including leaves, fruit and branches. One of the most damaging is the fruit rot phase known as black rot which causes the fruit of apples and pears to rot before harvest and in storage. The disease can cause latent infections of these fruits which do not become apparent until after harvest. The first visible symptoms of latent fruit infection are small black lesions (2-4 mm diam.) which are slightly sunken with a corky texture. These black lesions do not enlarge further and only give rise to a rapidly progressing pale brown rot 2-3 weeks preceding harvest. The active stage of the fruit rot can be seen in the orchard and is characterised by rot that has concentric zones of lighter and darker brown colours, later the rotted areas turn black. Fruits affected by this kind of brown rot are rapidly colonised within 3-5 days. The fungus also causes a distinctive leaf spot, known as frogeye spot. Leaf lesions are initially small, purple specks that enlarge to form spots 3 to 6 mm in diameter, these spots have light brown-to-grey centres which are surrounded by one or more darker rings of tissue and a purple border. Dark pycnidia of the fungus may develop in the centre of older leaf spots. Stem symptoms of D. seriata begin as slightly sunken, reddish-brown patches within the bark. These areas enlarge and darken to form cankers with sunken centres and raised margins. Cankers may also develop as a superficial roughening or cracking of the bark, especially at the margins, where the cankers girdle the twigs or branches a blight and dieback is seen. D. seriata is regarded as an important pathogen of apple in the USA (Stevens, 1933; Brown and Britton, 1986; Brown-Rytlewski and McManus, 2000) but as a weak secondary pathogen on the same host in the UK and New Zealand (Laundon, 1973).

On grapevines D. seriata is known to cause the death of spring buds, leaf chlorosis, fruit rot and trunk dieback, with brown, hard necrosis of the wood that appears as wedge-shaped necrosis in cross sections of the affected plant parts (van Niekerk et al., 2006; Urbez-Torres, 2011). Other symptoms include internal streaking and pith necrosis of wood, failure of graft union in young vines and cane bleaching (Urbez-Torres, 2011). 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; Urbez-Torres, 2011). Recently Urbez-Torres (2011) proposed the name ‘Botryosphaeria dieback’ to include the increasing number of Botryosphaeriaceous species besides D. seriata that have been associated with most of the symptoms and diseases above. Reports of the virulence of this pathogen on grapes varies with some artificial inoculation studies (Spagnolo et al., 2017; Pinto et al., 2018; Reis et al., 2019) suggesting that it is a weak pathogen to grapevine and possibly takes advantage of weak or stressed plants (Qiu et al., 2016). 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. It is also possible that in species with a broad host range, such as D. seriata, virulence of any given isolate may vary according to the host that is being attacked.

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).

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).

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).

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.

D. seriata has been implicated in causing economic damage to fruit crops, forestry and ornamental plants around the world. It is recognised as being one of the most prominent pathogens involved in grapevine trunk disease (GTD, or grapevine decline). This pathology can result in the death of adult plants and therefore it produces severe economic losses all around the world. The worldwide economic cost for the replacement of dead grapevines is roughly estimated to be more than 1.5 billion dollars per year (Hofstetter et al., 2012). In recent years, GTD has been increasing in importance as it is increasingly found affecting plants at a younger age being commonly reported in vineyards that are over 7-year-old (Díaz and LaTorre, 2013). According to a survey led by the French Directorate General for Food (DGAL) in 2012, nearly 13% of French vineyards were affected by trunk diseases (Grosman and Doublet, 2012). In 2014 these diseases lowered the French potential wine production by 13%, according to the agriculture ministry and French Wine Institute (IFV). The IFV estimate that GTD is costing France the equivalent of 1bn euros ($1.14bn) annually in lost wine production, and more than 100,000 hectares of vineyard was lost in 2014. In California USA, Eutypa dieback and Botryosphaeria canker were estimated to have caused over 260 million dollars of damage in reduced yields and increase production costs (Siebert, 2001).

D. seriata is also damaging to the production of apples and pears. In south-eastern USA, fruit losses of between 25 and 50% have been reported due to black rot (Brown and Britton, 1986). Similarly, since 2007 when D. seriata was first recorded on apples in the Lower Elbe region (northern Germany), annual crop losses of over 5% at harvest have been reported (Brockamp and Weber, 2014).

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.

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.