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© 2007 Plant Management Network.
Accepted for publication 15 January 2007. Published 9 May 2007.


Glomerella acutata on Highbush Blueberry (Vaccinium corymbosum L.) in Norway


V. Talgø, H. U. Aamot, G. M. Strømeng, S. S. Klemsdal, and A. Stensvand, Norwegian Institute for Agricultural and Environmental Research, Høgskoleveien 7, 1432 Ås, Norway


Corresponding author: Venche Talgø. venche.talgo@bioforsk.no


Talgø, V., Aamot, H. U., Strømeng, G. M., Klemsdal, S. S., and Stensvand, A. 2007. Glomerella acutata on highbush blueberry (Vaccinium corymbosum L.) in Norway. Online. Plant Health Progress doi:10.1094/PHP-2007-0509-01-RS.


Abstract

In August 2004, Glomerella acutata Guerber & Correll was detected on fruits from highbush blueberry (Vaccinium corymbosum L.) for the first time in Norway. Both the conidial (Colletotrichum acutatum J. H. Simmonds) and the ascigerous (G. acutata) stage developed on naturally infected blueberry fruits. Perithecia also readily formed on blueberries and strawberries inoculated with a culture from highbush blueberry, and on artificial, solid medium (both on strawberry leaf agar and potato dextrose agar). To our knowledge this is the first report worldwide of the teleomorph of Glomerella acutata on a naturally infected host.


Introduction

Highbush blueberry (Vaccinium corymbosum L.) is a minor crop in Norway, and is grown commercially only on approximately 30 ha. Climatic conditions for highbush blueberry are only suitable in coastal regions in southern Norway (10).

Anthracnose rot of blueberry is a problem worldwide, causing pre- and post-harvest rots (1,5,6,9). Fertile perithecia of the perfect (sexual/teleomorphic) stage of C. acutatum were discovered for the first time by Guerber and Correll (3), during crosses of self-sterile monoconidial strains of C. acutatum isolated from apple, blueberry, kiwi, pecan, strawberry, avocado, and papaya. The species was named Glomerella acutata Guerber & Correll.

Here we report the first occurrence of anthracnose in highbush blueberry in Norway and the in vivo discovery of the teleomorph of G. acutata.


Location, Sampling, and Symptoms

A grower in Vest-Agder County in southern Norway reported extensive damage on highbush blueberry fruits in August 2004. Damaged berries were collected from randomly-selected plants in a field with 500 bushes. A mixed sample of both ripe and unripe berries from the cvs. Toro, Duke, and Patriot were investigated. Symptoms included some berries with brown, sunken, dry spots and some shrivelled berries. In general, the berries were small, and callus had formed in the dry spots. The dry spots did not host any fungi after incubation, and there were no signs of insect damage. The grower reported that there had been a hailstorm at an early stage of fruit development and that might explain the dry spots.


Incubation, Isolation, and Identification

All incubation of fruit or other plant material described below took place in moist chambers (sealed plastic boxes with 100% RH) at room temperature. After three days of incubation of the plant material from Vest-Agder, many berries were covered with gray mold (Botrytis cinerea), and such berries were removed from the incubation chamber. Two weeks after the incubation started, a few berries were partly covered with orange, conidial spore masses of C. acutatum emerging from densely situated acervuli.

The fungus was isolated on acidified potato dextrose agar (PDA) by transferring conidia from a naturally infected berry to the agar with a sterile inoculating needle. See Table 1 for morphological details. According to EPPO (2), conidial spores from C. acutatum should be in the range 8-16 (20) × 2.5-5 µm. Thus, the size of the conidia we initially found on the incubated fruit were in the upper range. Conidia obtained after an inoculation test in August 2006 were smaller (see Table 1). Setae were never observed in acervuli on berries.


Table 1. Characteristics (signs) of the imperfect (conidial) and perfect (ascigerous) stage of Glomerella acutata from highbush blueberry.

         Imperfect stage Perfect stage
On naturally infected highbush blueberry fruit Orange, slimy conidial spore masses, with conidia measuring 15.0-(18.0)-22.5 × 4.5-(4.9)-5.8 µm. No mycelium observed. Conidia were hyaline, straight, cylindrical, some slightly constricted in the middle, aseptate, and mainly pointed in one end and rounded in the other end (96%). The remaining 4% were rounded in both ends. Dark brown perithecia (not further described).
On inoculated, detached highbush blueberry fruit Pinkish mycelium and/or orange, slimy conidial spore masses, with conidia measuring 5.6-(7.2)-9.6 × 1.6-(1.7)-1.9 µm. Twenty four, 72, and 4% of the conidia were pointed in one and rounded in the other end, pointed in both ends, or rounded in both ends, respectively. Dark brown perithecia measuring 210.0-(279.6)-390.0 × 115.0-(158.2)-190.0 µm, asci 52.5-(70.6)-95.0 × 7.5-(10.6)-12.5 µm, and ascospores 10.0-(13.6)-16.3 × 4.3-(5.0)-5.5 µm. Ascospores were slightly curved, and 30, 36, and 34% of the ascospores were pointed in one end and rounded in the other end, pointed in both ends, or rounded in both ends, respectively. In saturated air, orange ascosporic masses oozed out from perithecia and were much drier than the conidial spore masses.
On inoculated, detached strawberry fruit Pinkish mycelium. No conidia observed. Dark brown perithecia (not further described).
On potato

dextrose agar

Pinkish mycelium, no formation of acervuli and/or conidia. Colony growth was 63 mm and 47 mm after 7 days at 25°C and 29°C, respectively. A few dark brown perithecia (not further described).
On strawberry leaf agar Mycelium not visible to the naked eye, no formation of acervuli and/or conidia. Abundance of perithecia on strawberry leaf disks. Perithecia measuring 162.5-(217.5)-337.5 × 97.5(147.9)-207.5 µm, asci 60.0-(68.3)-77.5 × 10.0-(10.4)-12.5 µm, and ascospores 10.0-(12.6)-15.0 x 4.5-(5.1)-7.5 µm.

After five weeks of incubation of the infected berry, numerous perithecia (small black spots) of G. acutata had emerged in and between the conidial spore masses (Fig. 1). The isolate obtained from the conidia was self-fertile, and perithecia were also readily produced on potato dextrose agar (PDA) and strawberry leaf agar (SLA) (Fig. 2). SLA was prepared as described by Gunnell and Gubler (4), but we used autoclaved strawberry leaf discs (19 mm diameter) instead of dried, sterilized leaf pieces. For each Petri dish three leaf discs were floated on molten water agar.


 

Fig. 1. Fruit of highbush blueberry infected by Glomerella acutata five weeks after start of incubation in saturated air at room temperature. The orange conidial spore mass is the imperfect stage of the fungus (Colletotrichum acutatum) and the black spots in and between the conidial spore masses are perithecia (the perfect stage). Photo by E. Fløistad.

 

Fig. 2. Glomerella acutata readily (2 to 3 weeks after subculturing) formed perithecia on strawberry leaf agar (SLA). As seen here, perithecia have formed especially densely on the leaf disk, but were also found scattered in the agar (dark spots). Photo by V. Talgø.


To our knowledge, development of perithecia on a naturally infected host has never been observed elsewhere, although it was obtained in vitro by crossing different self-sterile strains of C. acutatum (3).

No formation of acervuli or conidia was observed on agar. A single spore isolate was made by floating a PDA culture with sterile water. The ascospore suspension obtained was spread onto water agar (WA). After 24-h incubation at room temperature, a single spore was obtained from the WA and transferred to PDA. The colony was sub-cultured on PDA and used for further testing.

A previously-described PCR test (7) was run both on an infected berry and on a single spore culture, and both proved positive for G. acutata.

The culture obtained from highbush blueberry produced mycelium with a distinct pinkish colour different from the greyish colour of C. acutatum isolates we usually obtained from strawberry (Fig. 3). Colony growth is described in Table 1. The pinkish colour of the mycelium has also been observed on C. acutatum cultures from highbush blueberry in Slovenia (5) and The Netherlands (J. Meffet, Dutch Plant Protection Service, Geertjesweg 15, Wageningen, The Netherlands, personal communication). From Canada it is reported that colonies of C. acutatum isolated from highbush blueberry appear bright pink to orange when viewed from the underside of the Petri dish (9).


 

Fig. 3. Cultures of Colletotrichum acutatum originated from strawberry (Fragaria × ananassa) (left) and Glomerella acutata from highbush blueberry (right). In the strawberry isolate, orange conidial spore masses are easily visible in the center. Conidia were never found in the culture from highbush blueberry. Photo by V. Talgø.

 

Perithecia on SLA were dark brown (Fig. 4). Morphological characteristics for perithecia, asci, and ascospores are given in Table 1. Asci (Fig. 5) bore eight ascospores. Ascospores on highbush blueberry in Norway were in the lower range compared to the spore size observed by Guerber and Correll (3) after crossing self sterile strains of C. acutatum, but they do approximately match the ascospore size they obtained when crossing self sterile strains from apple from the USA and kiwi from New Zealand (mean 12.2 × 5.0 µm).


 

Fig. 4. Perithecia from a culture of Glomerella acutata isolated from highbush blueberry and grown on strawberry leaf agar (SLA). Photo by H. U. Aamot.

 

Fig. 5. Asci and ascospores (stained with lacto fuchsin) of Glomerella acutata from perithecia that developed on a culture from highbush blueberry. Photo by H. U. Aamot.


Inoculation Tests

Koch’s postulates were fulfilled by inoculating attached green berries on a potted highbush blueberry plant (cv. Goldtraube 71). Small droplets (10 µl) of spore suspension (approximately 200 spores per drop) or agarplugs with mycelium (4 mm²) were placed on the berries that were either wounded or not wounded. A sterile inoculation needle was forced through the drops or agarplugs and into the tissue when wounding. The plant was covered with polythene bags for 24 h and thereafter kept for 2 months in a growth chamber. No symptoms developed. The berries were then harvested and incubated. After four weeks in saturated air at room temperature, one wounded berry inoculated with spore suspension was covered with conidia. G. acutata was readily reisolated from the infected berry, and the morphological characters developed in culture as described above. The reisolated fungus and the infected berry were tested with PCR and both proved positive for G. acutata. Inoculations of young shoots and leaves of the same plant and a non-inoculated control plant did not develop symptoms of the disease after two months in growth camber and an additional 4 weeks incubation of detached plant parts in saturated air.

The culture used for the inoculation test had been sub-cultured a number of times since it was first isolated in 2004. It is known that virulence can get lost during such a process. The photo of the highbush blueberry sub-culture in Fig. 3 was taken in June 2006, almost two years after the original isolation was done, and the mycelium was less pinkish than the original culture. Mycelium turning white during repeated subculturing can indicate loss of virulence. This was noted with C. kahawae on coffee berries and might explain the low infection rate in the inoculation test [Roger Cook, consultant plant pathologist and secretary of European Mycological Network (EMN), personal communication].

To test the culture’s ability to form perithecia on berries, 60 detached, ripe highbush blueberries (cultivar unknown) were inoculated in June 2006. Ten berries were kept as control (not inoculated). Droplets of an ascospore suspension (105 spores/ml) were placed on both wounded (with a sterile inoculation needle) and non-wounded berries. After a few days many berries were overgrown by B. cinerea, Trichothecium roseum, or Aspergillus niger and had to be discarded. After four weeks of incubation, acervuli containing conidia and perithecia containing ascospores of G. acutata had formed on three of the berries.

Another inoculation test was run in August 2006. Detached, immature berries of cv. Patriot were inoculated (115 fruits inoculated, 30 fruits non-inoculated controls) by wounding the skin with a sterile inoculation needle and placing mycelium in the wounds. After 4 weeks incubation, perithecia were detected on 35 (30%) of the inoculated berries (Fig. 6 and 7). None of the control berries showed symptoms of anthracnose. The asexual stage was produced on 13 berries (10%) of the inoculated berries. Only one berry had both the sexual and asexual stage present. Morphological characteristics for perithecia, asci, and ascospores are given in Table 1. Ascopore masses oozing out from perithecia looked different from conidial spore masses from acervuli. The colour was similar (orange), but the ascosporic masses (Fig. 7) appeared drier and less slimy than the conidial masses (Fig. 1). Reisolations were carried out from ascospores and pinkish mycelium that formed on some berries, and resulted in colonies identical to the original culture.


 

Fig. 6. Highbush blueberry fruit covered with perithecia of Glomerella acutata four weeks after inoculation with a G. acutata isolate from highbush blueberry. Photo by H. U. Aamot.

 

 

Fig. 7. Ascospore masses of Glomerella acutata oozing out from perithecia on the surface of a highbush blueberry fruit (cv. Patriot) inoculated with G. acutata. Photo by V. Talgø.

 

Fig. 8. Conidia of Glomerella acutata that developed after inoculating immature fruits from highbush blueberry (cv. Patriot) in August 2006. Photo by H. U. Aamot.


Ten days after inoculation, typical anthracnose (blackspot) symptoms on a strawberry inoculated with the G. acutata isolate from highbush blueberries had developed (Fig. 9). When left in a moist chamber for an additional week, mycelium covered the black, sunken lesion and the characteristic, pinkish mycelium and perithecia developed (Fig. 10).


 

Fig. 9. Anthracnose on a strawberry ten days after inoculation with Glomerella acutata originating from highbush blueberry. Photo by H. U. Aamot.

 

Fig. 10. A week after typical blackspot symptoms (see Fig. 9) developed on a strawberry inoculated with Glomerella acutata from highbush blueberry, a pinkish mycelium and perithecia of G. acutata covered the black, sunken lesion. Photo by V. Talgø.


The conidial stage (C. acutatum) of the pathogen has also been isolated from a number of other plant species in our country: two Cotoneaster species; blackberry (Rubus fruticosus); raspberry (R. idaeus); holly (Ilex aquifolium); northern dock (Rumex longifolius); common nettle (Urtica dioica); and all fruit crops grown in Norway (apple, pear, plum, and sweet and sour cherries) (8).


Current situation

During the summer and fall of 2006, a number of samples of fruits, leaves, and twigs of different varieties of highbush blueberry (cvs. Bluecrop, Chippewa, Spartan, St. Cloud, Toro, Polaris, and some mixed samples) were forwarded to us by the grower we received the positive sample of G. acutata from in 2004. The investigation did not yield any new findings of G. acutata, and we have not received samples containing the fungus from other growers. Hence, we believe that anthracnose is of minor importance for the highbush blueberry production in our country.


Acknowledgments

We want to thank Roger Cook for sharing his time with us on this matter. We also want to thank Helen M. Singh and Grete Lund at the Norwegian Institute for Agricultural and Environmental Research for valuable technical assistance, and the grower Tellef Rislaa for very good cooperation.


Literature Cited

1. De Marsay, A. 2005. Anthracnose fruit rot of highbush blueberry: Biology and epidemiology. Ph.D. theses, Rutgers, The State Univ. of New Jersey, New Brunswick, NJ.

2. EPPO. 2004. Glomerella acutata. OEPP/EPPO Bull. 34:193-199.

3. Guerber, J. E., and Correll, J. C. 2001. Characterization of Glomerella acutata, the teleomorph of Colletotrichum acutatum. Mycologica 93:216-229.

4. Gunnell, P. S., and Gubler, W. D. 1992. Taxonomy and morphology of Colletotrichum species pathogenic to strawberry. Mycologia 84:157-165.

5. Munda, A., and Metka, Z. 2006. Diseases of high-bush blueberry in integrated production plantations in Slovenia. 5th IOBC meeting of Integrated Plant Prot. in Fruit Crops Working Group (subgroup Soft Fruits), Stavanger, Norway, 5-7 October 2005. IOBCwprs Bull. (in press).

6. Schilder, A. 2006. Anthracnose (ripe rot). Colletotrichum acutatum (fungus). Online. Michigan Blueberry Facts. Michigan State Univ., East Lansing, MI.

7. Sreenivasaprasad, S., Sharada, K., Brown, A. E., and Mills, P. R. 1996. PCR-based detection of Colletotrichum acutatum on strawberry. Plant Path. 45:650-655.

8. Stensvand, A., Talgø, V., Strømeng, G. M., Aamot, H. U., Børve, J., Sletten, A., and Klemsdal, S. S. 2006. Colletotrichum acutatum in Norwegian strawberry production and sources of potential inoculum in and around strawberry fields. 5th IOBC meeting of Working Group "Integrated Plant Protection in Fruit Crops" (Subgroup "Soft Fruits"), Stavanger, Norway, 5-7 October 2005. IOBCwprs Bull. (in press).

9. Verma, N., MacDonald, L., and Punja, Z. K. 2006. Inoculum prevalence, host infection and biological control of Colletotrichum acutatum: causal agent of blueberry anthracnose in British Columbia. Plant Path. 55:442-450.

10. Vestrheim, S., Haffner, K., and Grønnerød, K. 1997. Highbush blueberry production and research in Norway. Acta Hort. 446:177-180.