A fruit rot resembling Gloeosporium infections but appearing on fruits prior to harvest was

noticed in organic apple orchards in Holland, Belgium and Northern Germany in 2007.

Infections were most commonly observed on ‘Elstar’, but other cultivars were also

affected. Fruit colonisation progressed in two steps, whereby a latent stage of sunken

black lesions in immature fruits gave rise to a rapidly spreading firm brown rot upon fruit

ripening. Isolation experiments from both stages consistently yielded a single species of

fungus identified as Diplodia seriata, formerly known under the teleomorph name

Botryosphaeria obtusa. Lesions of D. seriata were also seen on leaves as necrotic light

brown spots surrounded by a purple halo, and occasionally on small twigs as cankers.

Fruit mummies on apple twigs were heavily colonised by D. seriata and are thus likely to

carry inoculum for fruit infections during late summer or in the following growing season

Maxin, Peter

Trapman, Marc

Weber, Roland W. S.

English

Organic Eprints

177Diplodia seriata, cause of black fruit rot in organically grown apples in Holland, Belgium and Northern Germany Marc Trapman1, Peter Maxin2, Roland W. S. Weber3,*AbstractA fruit rot resembling Gloeosporium infections but appearing on fruits prior to harvest was noticed in organic apple orchards in Holland, Belgium and Northern Germany in 2007. Infections  were  most  commonly  observed  on  ‘Elstar’,  but  other  cultivars  were  also affected.  Fruit  colonisation  progressed  in  two  steps,  whereby  a  latent  stage  of  sunken black lesions in immature fruits gave rise to a rapidly spreading firm brown rot upon fruit ripening. Isolation experiments from both stages consistently yielded a single species of fungus  identified  as  Diplodia  seriata,  formerly  known  under  the  teleomorph  name Botryosphaeria obtusa. Lesions of D. seriata were also seen on leaves as necrotic light brown spots surrounded by a purple halo, and occasionally on small twigs as cankers. Fruit mummies on apple twigs were heavily colonised by D. seriata and are thus likely to carry inoculum for fruit infections during late summer or in the following growing season.Keywords: Botryosphaeria obtusa, Diplodia seriata, apple, black rot, fruit mummies IntroductionIn August and September 2007 an unusual fruit rot was noticed on ripening apples in Belgium,  the  Netherlands  and  in  the  Lower  Elbe  Valley  of  Northern  Germany.  Whilst ‘Elstar’  was  most  commonly  affected,  the  rot  was  also  seen  on  several  other  apple varieties  including  ‘Ingrid  Marie’,  ‘Dalinco’,  ‘Dalinbel’,  ‘Jonagold’  and  ‘Gerlinde’.  The disease  was  observed  only  in  organically  managed  orchards  where  it  appeared  to progress in two stages. The first visible symptoms were small black lesions (2-4 mm diam. and in depth) which were slightly sunken and had a somewhat corky texture upon cutting. These black lesions did not enlarge further and were regarded as a dormant stage of infection because they gave rise to a rapidly progressing pale brown rot only during the 2-3 weeks preceding harvest (Fig. 1). The active fruit rot showed a concentric zonation of lighter and darker brown colours (Figs. 1 and 6). Fruits affected by this kind of brown rot were entirely colonised by it within 3-5 d. In contrast, no brown rot arose from the black lesions when infected apples were harvested and stored at low temperatures (2-4 °C) for up to 6 weeks, although the rot appeared within a few days of incubating these stored apples at room temperature. Leaves of infected trees showed a purple halo surrounding necrotic tan-coloured spots, some of which carried pycnidial primordia (Fig. 2). Severe leaf infections led to premature defoliation soon after harvest and were seen predominantly on trees also carrying infected fruits. Less severe leaf infections were observed on trees up to 100 m away from the infection focus. In severely affected orchards, up to 25% of the harvest was lost. Since we had not previously observed this disease, we isolated and identified the causal agent._________________________1 Bio Fruit Advies, Dorpsstraat 32, 4111 KT Zoelmond, Netherlands 2 KÖN and 3 OVA, Obstbau Versuchs- und Beratungszentrum (OVB) Jork, Moorende 53, 21635 Jork, Germany * Corresponding author: roland.weber@lwk-niedersachsen.de Archived at http://orgprints.org/13668/178Figures 1-2. Obvious symptoms of D. seriata on ‘Gerlinde’. Fig. 1: Successive stages of fruit rot from left to right. Fig. 2: Leaf infections of the ‘frog-eye’ type in which a pale brown necrotic centre is surrounded by a reddish-purple halo.  Material and methods Fruits showing dormant black lesions and/or spreading brown rot were surface-sterilised by wiping with tissue paper soaked in 70% ethanol. Small tissue pieces (2-4 mm3) were cut from the advancing margin of infections with a sterile scalpel and were transferred to 2% malt extract agar (MEA) augmented with penicillin G and streptomycin sulphate (each at 200 mg l-1). For initiation of pycnidial development and spore production, isolates were subcultured onto tap-water agar (TWA) bearing small (5 mm) lengths of Pinus needles (Slippers et al., 2007). Because of its distinctive spores, it was possible subsequently to identify the fungus by direct microscopic analysis of sporulating lesions taken from other infected host organs, especially twig cankers and fruit mummies. ResultsIsolation of D. seriata. – On MEA, one and the same species of fungus was consistently isolated from dormant black lesions as well as actively spreading brown rot infections. Whereas rapid growth ensued within 24 h of plating out brown rot samples, with black lesions a lag time of 48-72 h was observed before mycelium became visible. Colonies on MEA  were  initially  hyaline,  but  produced  dark  green  melanised  hyphae  and  pycnidium initials within 7 d incubation at room temperature (20-22 °C). Mature pycnidia with typical conidia were observed on TWA with pine needles after 14-21 d incubation.12Archived at http://orgprints.org/13668/179Description of D. seriata. – Conidia were ellipsoid to cylindrical (22-25 × 9.5-11.0 µm) with a distinct basal scar. They had conspicuously thick (>0.5 µm) melanised cell walls and contained dense cytoplasmic deposits of lipid droplets which made it impossible to state with certainty whether the walls were smooth or slightly roughened. Exuded as aseptate and  fully  pigmented  spores,  conidia  were  occasionally  observed  to  form  one  central transverse septum at a later stage (Fig. 3). Identification as Diplodia seriata De Not. was unequivocal  on  the  basis  of  the  descriptions  given  by  Punithalingam  &  Waller  (1973), Jones  &  Aldwinckle  (1990)  and  Phillips  et  al.  (2007).  Pale  brown  leaf  spots  (Fig.  2) showing the typical ‘frog-eye’ appearance described by Jones & Aldwinckle (1990) were also assigned to infections by D. seriata.Distribution  of  D.  seriata.  –  On  the  basis  of  its  conspicuous  conidia,  we  were  able  to examine trees from an orchard heavily infested with fruit rot (‘Gerlinde’; Neuenfelde, Altes Land, Germany) for the presence of D. seriata on other host organs. Whereas cankers were only rarely observed on two-year-old twigs (Fig. 4), infections of fruit mummies were extremely  abundant  (Fig.  5).  Of  100  mummies  collected  from  10  trees  with  fruit  rot symptoms, 97 were heavily colonised by D. seriata. Similarly dense colonisation of fruit mummies was observed on material from other Northern German orchards as well as from Belgium and Holland. We were able to confirm D. seriata in fruit rots from the following samples provided by organic fruit farmers: (1) Northern Germany, on ‘Dalinbel’, ‘Elstar’, ‘Gerlinde’,  ‘Holsteiner  Cox’  and  ‘Ingrid  Marie’  in  Hove,  Neuenschleuse,  Estebrügge, Neuenfelde (Altes Land, Lower  Elbe Valley); (2) Netherlands,  on ‘Dalinco’, ‘Elstar’ and ‘Jonagold’ in Beemster and ‘s-Hertogenbosch; (3) Belgium, on ‘Elstar’ in Reinrode.  Figures 3-5. Further diagnostic features of D. seriata. Fig. 3: Conidia from colonised fruit mummies (top  row)  and  from  an  agar  culture  (bottom).  Fig.  4:  Canker  on  a  two-year-old  ‘Gerlinde’  twig. Infection probably occurred through the pruning wound (arrow). Fig. 5: Infected fruit mummy from a ‘Gerlinde’ tree.45310 µm Figures 1-2. Obvious symptoms of D. seriata on ‘Gerlinde’. Fig. 1: Successive stages of fruit rot from left to right. Fig. 2: Leaf infections of the ‘frog-eye’ type in which a pale brown necrotic centre is surrounded by a reddish-purple halo.  Material and methods Fruits showing dormant black lesions and/or spreading brown rot were surface-sterilised by wiping with tissue paper soaked in 70% ethanol. Small tissue pieces (2-4 mm3) were cut from the advancing margin of infections with a sterile scalpel and were transferred to 2% malt extract agar (MEA) augmented with penicillin G and streptomycin sulphate (each at 200 mg l-1). For initiation of pycnidial development and spore production, isolates were subcultured onto tap-water agar (TWA) bearing small (5 mm) lengths of Pinus needles (Slippers et al., 2007). Because of its distinctive spores, it was possible subsequently to identify the fungus by direct microscopic analysis of sporulating lesions taken from other infected host organs, especially twig cankers and fruit mummies. ResultsIsolation of D. seriata. – On MEA, one and the same species of fungus was consistently isolated from dormant black lesions as well as actively spreading brown rot infections. Whereas rapid growth ensued within 24 h of plating out brown rot samples, with black lesions a lag time of 48-72 h was observed before mycelium became visible. Colonies on MEA  were  initially  hyaline,  but  produced  dark  green  melanised  hyphae  and  pycnidium initials within 7 d incubation at room temperature (20-22 °C). Mature pycnidia with typical conidia were observed on TWA with pine needles after 14-21 d incubation.12Archived at http://orgprints.org/13668/180DiscussionDiplodia  seriata,  better  known  in  the  literature  as  Botryosphaeria  obtusa  (Schwein.)Shoemaker, is readily recognised by its large, ellipsoidal to cylindrical conidia which are aseptate but fully pigmented while still within the pycnidium, and it can be distinguished on that basis from other Botryosphaeria spp. Conidia of B. stevensii Shoemaker (anamorph Diplodia mutila (Fr.) Mont.) become septate early in their development, but melanisation is delayed until they are outside the pycnidium; whilst B. dothidea (Moug.:Fr.) Ces. & De Not. (anamorph Fusicoccum aesculi  Corda) has conidia which remain hyaline and aseptate even  at  maturity  (Jones  &  Aldwinckle,  1990;  Phillips,  2007).  Despite  these  clear-cut differences, the taxonomic placement of D. seriata has been in a state of confusion which persists to the present day. Slippers  et al. (2007) have shown that this species is not closely related to currently recognised Botryosphaeria spp. such as B. stevensii and B.dothidea, and they have proposed that it should no longer be called B. obtusa. For the moment,  the  best  available  name  seems  to  be  D.  seriata  (Phillips  et  al.,  2007).  The circumscription of this fungus is further complicated by the likely existence of numerous strains or cryptic species (Phillips, 2007).  Our observation of the existence of a latent phase of D. seriata in apple fruits agrees with several reports on B. dothidea which can infect apples within 7 weeks of petal fall but shows a delay of brown rot until the defence system weakens during fruit ripening (Parker & Sutton, 1993; Kim et al., 2001). This aspect of the infection biology of D. seriata and B.dothidea is very similar also to Gloeosporium spp. causing storage rots of apples. Such similarities extend to the colonisation of fruit mummies and the bark of trees as niches for overwintering. We were astonished to find that nearly all mummies on trees with fruit rot symptoms were infected by D. seriata. These mummies (1-2 cm diam.) partly originated from aborted fruits of the current growing season, and partly represented overwintered structures from the previous year. We regard them as the key to the success of D. seriataas an apple pathogen (Fig. 6), and it is probably no coincidence that varieties with a strong tendency to retain their mummies (e.g. ‘Elstar’ or ‘Gerlinde’) were more frequently infected than others. Extended warm and wet periods in Northern Europe in May, June and July 2007 would have been favourable for fruit infections from colonised mummies, rainsplash being the main dispersal mechanism for D. seriata conidia (Arauz & Sutton, 1989). We have not (yet) found D. seriata in orchards under integrated pest management (IPM) in Northern Germany. This could, of course, be coincidental, i.e. due to chance observations by organic farmers but not their conventional colleagues. More likely, the latter may have achieved collateral control of D. seriata by fungicide treatments against Gloeosporium spp. or other pathogenic fungi during the growing season. The near-absence of sooty blotch disease from IPM orchards in Northern Germany probably has a similar explanation. In organic fruit production, the removal of fruit mummies from the trees, although labour-intensive, may be a good way to control D. seriata and to retard its further spread. In the same  instance,  this  would  contribute  to  the  control  of  several  other  fungal  pathogens, notably Gloeosporium and Fusarium spp. which can co-colonise fruit mummies infected by D. seriata (Weber et al., 2008). However, such hygiene measures should take into account that D.  seriata  has  numerous  alternative  hosts,  including  quince,  pear,  peach,  plum, grapevine and many other woody plants (Punithalingam & Waller, 1973; Phillips et al.,2007;  Slippers  et  al.,  2007).  When  considering  other  plants  as  alternative  hosts,  one should also test for possible symptomless (endophytic) colonisation of twigs by D. seriata(Slippers & Wingfield, 2007). Further work on the spread, infection biology and control of D. seriata in Northern European apple orchards is therefore urgently required before more secure control measures can be recommended. Archived at http://orgprints.org/13668/181Figure 6: Fruit mummies were the likely source of infection for black lesions and a spreading brown rot (arrow) of Diplodia seriata.ReferencesArauz,  L.F.  &  Sutton,  T.B.  (1989).  Temperature  and  wetness  duration  requirements  for  apple infection by Botryosphaeria obtusa. Phytopathology 79: 440-444. Jones, A.L. & Aldwinckle, H.S., eds. (1990). Compendium of Apple and Pear Diseases. St. Paul, USA: APS Press.  Kim, K.W., Park, E.W., Kim, Y.H., Ahn, K.-K., Kim, P.G. & Kim, K.S. (2001). Latency- and defense-related ultrastructural characteristics of apple fruit tissues infected with Botryosphaeria dothidea. Phytopathology 91: 165-172. Parker, K.C. & Sutton, T.B. (1993). Susceptibility of apple fruit to Botryosphaeria dothidea and isolate variation. Plant Disease 77: 385-389. Phillips, A.J.L. (2007). The Botryosphaeria Site. http://www.crem.fct.unl.pt/botryosphaeria_site  Phillips, A.J.L., Crous, P.W. & Alves, A. (2007). Diplodia seriata, the anamorph of “Botryosphaeria”obtusa. Fungal Diversity 25: 141-155. Punithalingam, E. & Waller, J.M. (1973). Botryosphaeria obtusa. CMI Descriptions of Pathogenic Fungi and Bacteria 394.Slippers, B. & Wingfield, M.J. (2007). Botryosphaeriaceae as endophytes and latent pathogens of woody plants: diversity, ecology and impact. Fungal Biology Reviews 21: 90-106. Slippers,  B.,  Smit,  W.A.,  Crous,  P.W.,  Coutinho,  T.A.  &  Wingfield,  B.D.  (2007).  Taxonomy, phylogeny and identification of Botryosphaeriaceae associated with pome and stone fruit trees in South Africa and other regions of the world. Plant Pathology 56: 128-139. Weber, R.W.S., Maxin, P. & Trapman, M. (2008).  Diplodia seriata als Ursache der schwarzen Sommerfäule im ökologischen Apfelanbau Norddeutschlands. Mitteilungen des OVR des Alten Landes 63 (in press). Archived at http://orgprints.org/13668/

Diplodia seriata, cause of black fruit rot in organically grown apples in Holland, Belgium and Northern Germany

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