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Survey of Blueberry Diseases in Tasmania
Ziqing Yuan, Dean Metcalf, Michele Buntain, Wayne Williams and
Changyou Pan
Biosecurity & Product Integrity Plant Health Branch
May 2009
Acknowledgments Dr Ross Corkrey of Tasmanian Institute of Agriculture Research is thanked for the advice on the statistically based sampling. Survey plan was approved by Margaret Williams, the Branch Manager prior to the field surveys. Thanks are due to M. Williams for her consistent support in preparation of the report and to Cindy Hanson of Biosecurity Policy Branch for valuable comments on version 1. Dr Morag Glen of CSIRO, Sustainable Ecosystems provided valuable assistance in conducting DNA sequence analyses for Colletotrichum species. All the farmers and owners of orchards who participated in the surveys are also gratefully acknowledged.
SUMMARY Two comprehensive surveys designed specifically to determine Tasmanian area freedom from blueberry anthracnose were conducted in February-March and October-November 2008 across Tasmanian blueberry industry areas. Following the national disease survey (sampling) guidelines outlined by Plant Health Australia, nineteen commercial blueberry farms were visited, representing 95% of total commercial blueberry operations in Tasmania. A total of 3,332 plants were inspected for anthracnose and other diseases including blueberry rust, during each survey. About 260 plant specimens (leaves and twigs/stems) and 16,284 berries were sampled during the surveys. Blueberry rust caused by Pucciniastrum vaccinii and anthracnose caused by Colletotrichum acutatum and Colletotrichum gloeosporioides occurring in mainland Australia were not detected in Tasmania. Two different Colletotrichum species were detected respectively on a single twig of a dead plant in an orchard and on cuttings in a nursery. The species from the dead plant was tentatively identified as C. aff. acutatum and the one from cuttings as C. aff. gloeosporioides. Our morphological study and DNA sequence analysis have indicated that they are different from all three Colletotrichum species (races) detected on blueberry fruits imported from New South Wales. It was determined that:
• The Tasmanian C. aff. acutatum is genetically different from C. acutatum (race 1 & 2) originating from NSW.
• C. aff. acutatum is genetically related or probably conspecific to the isolate of C. acutatum from tomato in Tasmania. C. aff. acutatum is a non host-specific, saprophytic species, which is not established as a pathogen on blueberry in Tasmania.
• C. gloeosporioides from fruits originating from NSW is a true C. gloeosporioides. The Tasmanian C. aff. gloeosporioides is morphologically and genetically different from C. gloeosporioides from NSW.
• C. aff. gloeosporioides is not a true C. gloeosporioides and is possibly conspecific to C. kahawae. C. aff. gloeosporioides is tentatively identified as C. aff. kahawae, but is referred to in the report as C. aff. gloeosporioides.
• C. aff. gloeosporioides could be a weak pathogen on blueberry but has not been encountered outside the nursery where it was recovered on four occasions. Trace back and trace forward investigations failed to find any incidence of C. aff. gloeosporioides.
• No anthracnose was found on fruits during inspection of 16,284 fruits, either freshly harvested or after incubation at different temperatures.
This is in contrast to the high incidence of anthracnose fruit rot in the fruits from NSW detected in 2007.
• The original isolate of C. lupini from lupins in Tasmania, during the incursion in 2004, is unrelated to all Colletotrichum species on blueberry and other hosts and appears to be as host-specific to lupins as those from WA and NSW.
On the above basis, it is concluded that Tasmania is free from C. acutatum and C. gloesporioides races causing anthracnose. This survey has shown that no severe disease problems are present on leaves, stems and fruits in commercial blueberry orchards in Tasmania. Twig dieback or “winter chill” was the most commonly encountered symptom and a wide range of fungal species was isolated from dieback twigs. The phenomenon is widely considered to be physiological. Although several of the fungi obtained in this survey are known to be pathogenic or belong to genera which contain recognised pathogens, e.g. Botryosphaeria sp., Coniothyrium sp., Pestalotiopsis spp. and Seimatosporium spp., except for Pestalotiopsis spp., they were only encountered occasionally and impact of damage is currently minor. Our preliminary morphological studies have shown a number of fungi encountered during the survey are new records for Australia or new to Tasmania. Further taxonomic studies on these fungal species by other fungal taxonomic experts are needed to confirm the identifications. New records of fungal species identified during the first survey were:
• Alternaria tenuissima, recorded on blueberry for the first time in Australia;
• Mycosphaerella sp, a new record on blueberry in Tasmania;
• Harknessia aff. globosa, this is the first time that a Harknessia species is found on blueberry;
• Seimatosporium aubuti, the first record on blueberries in Australia and new record species for Tasmania;
• Pestalotiopsis spp., three (arguably four) different species were encountered respectively on stems, twigs and cuttings showing canker or dieback symptoms and all are new records on blueberries for Tasmania.
New records of fungal species identified during the second survey were:
• Zetiasplozna thuemenii examined on the stem of a potted seedling imported from Victoria, a new record fungal species (genus) for Australia;
• Seimatosporium vaccinii, recorded on blueberry for the first time in Australia;
• Botrysphaeria sp. recorded on blueberry for the first time in Tasmania;
• Harknessia sp., (a different species from H. aff. globosa) recorded on blueberry for the first time;
• Valsa sp. (Cytospora sp.), recorded on blueberry for the first time in Tasmania.
New record of fungal species identified from nursery cuttings was:
• Colletotrichum aff. gloeosporioides (= C. aff. kahawae), probably a new record for Australia, but further investigation is required to confirm this status.
As well as demonstrating freedom from blueberry anthracnose, this survey has provided baseline information that will be invaluable for future fungal diagnosis and assessments of potential impact. Identification of more than 20 fungal species on blueberry for Tasmania during the relatively short period of the survey indicated how little is known about fungi on blueberry in Tasmania and possibly Australia, and their potential plant health significance.
INTRODUCTION During a routine 600 count inspection on 07/10/2007, Quarantine Tasmania intercepted blueberries imported from New South Wales (NSW) that were infected with two fungal diseases: blueberry rust (Figs 1-4, Plate 1) and anthracnose fruit rot (Figs 5-8, Plate 1). Both diseases were considered to be of highly significant quarantine concern to Tasmania. The rust was identified as Pucciniastrum vaccinii (G. Wint.) JØrst, which is a List A disease (a disease that does not occur at all in Tasmania) under the Plant Quarantine Act (Tas) 1997. Host material originating outside Tasmania is subject to specific import conditions and restrictions (DPIW, 2008). The disease has been reported on many species of Vaccinium from Asia, Europe and America (Farr et al., 2009). In Australia, it is recorded only on blueberry from NSW (APPD, 2009). Anthracnose fruit rot, also known as “ripe rot” is a major disease problem reducing yield and post-harvest fruit quality of highbush blueberry (Vaccinium corymbosum L.) in many blueberry growing areas in the world. Two species in the genus Colletotrichum, C. acutatum J. H. Simmonds and C. gloeosporioides (Penz.) Penz. & Sacc. are reported causing blueberry anthracnose globally (Caruso & Ramsdell, 1995). The pathogen primarily affects fruits, but can also attack all other aboveground parts of plants and develop various symptoms on leaves, twigs, stems and fruits. The disease is maintained from year to year in infected above ground parts (Yoshida et al., 2007). In Australia, C. acutatum is recorded on blueberry only in NSW and Victoria (VIC), and C. gloeosporioides in NSW and Western Australia (WA) (APPD, 2008). However, no systematic surveys appear to have been undertaken. The anthracnose fruit rot detected on the blueberries from NSW was initially attributed to C. gloeosporioides, but later C. acutatum (two races) was also isolated from the infected fruits. These three species (races) cause fruit rot symptoms that are slightly different in the appearance and the colour of the conidial masses that present on the surfaces of rotting fruits (Figs 6-8, Plate 1). Following the detection of the disease, a preliminary Import Risk Analysis (IRA) was undertaken (DPIW, 2007) and interim import requirements put in place. A month later, on 7/11/2007, four potted cuttings of blueberries were submitted from a local nursery to Plant Health Branch, DPIW for disease diagnosis. One of the dying stems, from which a Cylindrocarpon species was predominantly recovered on Pectin Agar (PA), was found to be covered with fruiting bodies of Glomerella sp. Its Colletotrichum anamorph was subsequently recovered from the stem along with Glomerella sp. on PA. Morphological examinations of this Colletototrichum on the cutting stem in Tasmania indicated a morphologically related species to C. gloeosporioides detected on blueberry fruits from NSW. The fungus was tentatively identified as C. aff. gloeosporioides.
Following the detection of that single incidence of C. aff. gloeosporioides on the cutting stem in Tasmania and consistent with Recommendation 4 of the preliminary IRA, a survey of blueberry fruits and plants in Tasmania was conducted in Feb-March, 2008 to determine the status of blueberry anthracnose and other diseases, including rust, in Tasmania. Since the seasonal conditions were so dry at that time, potentially affecting disease expression, the survey was repeated during late spring 2008 when conditions were expected to be more favourable to disease expression. This report details the methodology employed and the results obtained from both surveys.
METHODS Survey Design Fruits and plant materials including leaves, twigs and stems were inspected and sampled for diseases in accordance with the national guidelines for determining area freedom outlined by Plant Health Australia (Plant Health Australia, 2006). The survey method selected was to check for the presence of a disease in each of the disease niches (blueberry orchards or farms). As in the farming situation, blueberry plants are grouped by localities (growers) across Tasmania, and a survey scenario outlined by Plant Health Australia was employed (6.4.3.3.2.2.1, Plant Health Australia, 2006). Instead of 50% of farms which is suggested in the scenario, 95% of farms (19 of total 20) were visited during our surveys. Blueberry plants in home gardens were not surveyed. Determination of sample size for plants Sample size is usually determined from a given level of confidence (eg. 95%) and an assumed disease prevalence (the actual proportion of hosts that are infected) (eg. 0.1%-5%). The number of plants per property to be sampled was calculated as follows: As estimated in 2007, Tasmanian blueberry industry is about 30ha in size operated by about 20 growers (with around 1-3 ha each). Estimated total number of plants in Tasmania is: 2200 (numbers of plants/ha) x 30ha = 66,000. Since the total population of blueberry plants is relatively small and was considered as one population, the sample size (number of plants required to sample) to detect disease at an assumed prevalence of 0.1-0.5% with 95% confidence was calculated using the hypergeometric distribution according to the formula below: n = (1-(1 - α)^(1/d)) (N-d/2) + 1 Where: n = sample size
α = confidence level d = number of infected individuals in the population N = population size
The sample size determined from steps in the assumed prevalence range of interest are given in Table 1.
Table 1: Sample size (plant number) required to detect an infected individual plant in a given population of total of 66,000 plants, where between 0.1 and 5% of plants are believed to be infected, with 95% confidence; assuming the hypergeometric distribution.
Assumed prevalence Total number of plants = 66,000
5% 59
4% 74
3% 99
2% 149
1% 298
0.5% 596
0.1% 2928
As the aim was to detect at 0.1% prevalence or greater to test for area freedom, we needed to sample at least 2928 plants. The actual number of plants sampled was greater, adjusted to take account of certain potential deficiencies in the disease detection protocol. For instance, only a 90% chance of detection was achieved due to identification (test) failure (or false negatives including latent infections) of samples, meaning that a minimum number of 3253 (=2928/0.9) plants should be inspected for the detection of a disease with 95% confidence. Determination of sample size for fruits The probability of detection of anthracnose in fruits is affected by sample size. Since fruits were sampled predominantly in batches (punnets) in harvest areas and the size of the sample universe is relatively small, a beta-binomial-based sampling plan was used (Venette et al., 2002). The following formula was used to calculate the probability of detecting one or more infected fruit samples:
p(x>0) ≈ 1- (1+n θ)(-mf/ θ) Where: ө = aggregation coefficient (0 ≤ ө ≤ 1)* X = number of infected samples n = number of punnets or other containers m = number of fruits per punnet f = prevalence (*In this instance, an aggregation coefficient of 0.1 was used, assuming modest aggregation of diseased fruits.) Using this formula, to achieve a confidence of 95% and an assumed disease prevalence (0.1%), a minimum sample of 12,500 (=125 punnets x 100 fruits/punnet) berries was needed.
Plant Sampling To sample a plant, a symptom-based sampling method defined as “a sampling that involves searching each selected host, but only collecting a specimen from those with symptoms of the pest”. (Guideline Section 8.4.1.6, Plant Health Australia, 2006) was used for survey of plants, which meant plants with latent infections of any diseases could not be detected. Although there are reports that blueberry anthracnose pathogen, eg C. acutatum can be isolated from apparently healthy plants neighbouring diseased bushes (Hartung et al., 1981; Yoshida et al., 2007), the present survey was not aimed to detect diseases from symptomless plants, due to the resources available and the reduced chances of success. Since the blueberry plants are regularly arranged, systematic sampling was an appropriate practical choice of sampling method. Sampling for each of 19 orchards followed the procedures outlined below: 1. All rows were examined on foot. 2. For each orchard an estimate of the numbers of plants present was made and the proportion to be sampled determined.
3. Plants for detailed examination were found by choosing a starting point at random from plants 1-20 in any row and then every 10th or occasionally every 20th plant was chosen.
4. Selected plants were examined for disease symptoms on fruits (if applicable), leaves, twigs and stems (ca. 2-4 min. per plant).
5. Where presence of a pathogen was suspected, samples were taken to the Plant Health Laboratory at New Town in sealed and secure plastic bags for further examination and culturing tests.
Fruit Sampling A random sampling method (Guideline Section 8.4.1.4, Plant Health Australia, 2006) was used for the survey of fruits. The method is not symptom-based. The sampling was conducted in packing houses (areas) or from the plants inspected, if harvested fruits were not available. The number of fruits, in packs of punnets (ca. 100/punnet), to be sampled for each farm was determined according to a proportion of total plants to be inspected at the farm. The punnet samples varied with farms in a range of 2 to 27 punnets. In packing houses, fruits were randomly collected from fruits on the bench and packed in punnets. Fruits sampled from the field were picked randomly from the plants inspected for disease symptoms. An average of 10-25 berries per plant was picked. The plants for fruit samples were chosen starting from the first one to be inspected and continued until the number of punnets required for the farm was completed. The total number of berries sampled for each farm is listed in Table 2.
Field surveys Field surveys were conducted in two separate rounds; first around the blueberry harvest season from mid February to early March 2008 and then in spring, from October to November 2008. Out of total 20 commercial blueberry properties in Tasmania in early 2008, nineteen properties (representing 95% of the industry) were visited during each survey (Table 2). Table 2: Location of blueberry properties (farms) visited during the surveys.
Location Age of orchard Property size
(No. plants) No. of plants inspected
No. of berries collected
1 Frankford 25 + yr old 1000 98 464
2 Gravelly beach 25 + yr old 1600 116 160
3 Deloraine 1 and 2 yr old 900 88 --*
4 Western Creek 25 + yr old 7000 740 312
5 Lymington 25 + yr old 15000 700 2720
6 Oyster Cove 25 + yr old 1400 76 1678
7 Charlotte Cove 25 + yr old 1500 63 960
8 Lymington 15 + & 5yr old 2600 110 1010
9 Petchey’s Bay 25 + yr old 1100 45 670
10 Turners Marsh 25 + yr old 1600 68 1546
11 Lebrina 25 + yr old 6800 285 2286
12 Margate 25 yr + 2860 143 1983
13 Longley 2 to 4 yr old 3100 156 410
14 Glen Huon 5 yr old 960 48 168
15 Glen Huon 2 yr old 1160 62 --*
16 Petcheys Bay 25 + yr old 1000 101 200
17 Deep Bay, Sunday Hill 3, 4 yr & 7 yr old 1860 182 620
18 Howden 4 yr old 480 48 --*
19 Scottsdale 25 + yr old 2000 203 1107
Total 53920 3332 16,284
* No fruits were available for sampling.
Other diagnostic work on fungi associated with cutting death in nurseries Due to the detection of Colletotrichum aff. gloeosporioides from a cutting stem in a Tasmanian nursery (Nursery 1), an investigation of fungi associated with cutting death in nurseries was conducted, parallel to the disease survey in commercial orchards. Samples of dead or dying cuttings were collected from or submitted by Nursery 1 and another two (Nursery 2 and Nursery 3). They were examined and tested for C. aff. gloeosporioides and other fungal species.
Laboratory disease investigation All the samples collected from the field surveys were examined for the presence of plant diseases, including rust and anthracnose. Leaf and stem samples showing disease symptoms but no signs of pathogens present on the surfaces of diseased tissue were tested for fungal pathogens on artificial media. Isolations were made from well defined lesions on diseased plants. 10-15 small pieces of tissue for each sample were taken from the margin of lesions. Pieces were dipped in 95% ethanol and washed with sterile water, then surface-sterilised with 3% sodium hypochlorite for 2 minutes, and finally rinsed with sterile water. The surface-sterilised pieces were then put on pectin agar (PA) and incubated at 22°C in darkness. After 7-14 days the plates were inspected for fungal colonies. Comparison of conidial germination pattern and morphology of appressoria among Colletotrichum species was made by spore germination tests when fresh conidia were available. Fungal fruiting bodies associated with disease symptoms of the collected samples and those isolated from diseased plant tissue on media were identified to genus and species levels, based on the relevant literature for each fungal group. Fruit samples were examined under the dissecting microscope (x10 - x60) for anthracnose symptoms in the laboratory. To imitate storage temperature condition as for the fruits imported from NSW, each sample (punnet) was then divided into two portions with one incubated in a cool room (1-4 ºC) for up to 3 weeks and the other at high temperature (30 ºC) for 5-7 days to encourage disease symptoms to develop. Molecular characterization of Colletotrichum species isolated from blueberry Colletotrichum aff. gloeosporioides isolated from the local nursery cutting in 2007 and C. aff. acutatum isolated from a dead plant during the present survey were studied using molecular techniques by CSIRO, Sustainable Ecosystems based in Hobart (Glen, personal communication). Details of the methods, such as DNA extraction, PCR amplification and sequencing are not presented here but will be given in the manuscript for publication. Isolates of Colletotrichum species Five isolates from blueberry (three from NSW and two from Tasmania) were used in this study (Table 3). For comparison, one isolate of C. gloeosporioides (= C. lupini), originally isolated from lupins in 2004 when an incursion of lupin anthracnose occurred in Tasmania, was also included (Table 3).
Table 3: List of isolates of Colletotrichum species from blueberry and lupins
Code Species Host Origin
DM BB-167 C. aff. acutatum Blueberry plant Tasmania DM 119-07-A C. acutatum (race 1) Blueberry fruits NSW DM 119-07-B C. acutatum (race 2) Blueberry fruits NSW DM 134-07 C. aff. gloeosporioides Blueberry cuttings Tasmania DM 119-07-C C. gloeosporioides Blueberry fruits NSW DM 16-04* C. gloeosporioides (= C. lupini) Lupins Tasmania
* The isolate used here for comparison was the original isolate from lupins when the incursion of anthracnose occurred in Tasmania in 2004. The disease and the host plants from which the isolate was sourced have been eradicated with no further record of the pathogen in Tasmania.
DNA sequence analysis Nucleotide sequence analyses of two Tasmanian Colletotrichum species, along with C. acutatum (race 1 and 2), and C. gloeosporioides isolated from blueberry fruits imported from NSW and C. lupini isolated from lupins in Tasmania, were conducted in three separate assays: Assay 1:Sequence data on the region around the sixth intron of the β-tublin-2
gene were compared with DNA data of 63 Australian C. acutatum isolates and four C. gloeosporioides isolates published by Whitelaw-Weckert et al. (2007).
Assay 2:Sequence data on the region around the sixth intron of the β-tublin-2
gene were compared with DNA data of Colletotrichum species published by Whitelaw-Weckert et al. (2007) for Australian isolates and by others (Buhr & Dickman, 1994, Talhinhas et al., 2002, Vinnere et al., 2002 and Gene bank) for non Australian isolates.
Assay3:Sequence data on introns of glyceraldehyde-3-phosphate
dehydrogenase (GPD) were compared with DNA data of Colletotrichum species published by Liu et al. (2007), Mackenzie et al. (2007), Marcelino et al. (2008) and Templeton et al. (1992).
RESULTS Field survey and laboratory investigation No anthracnose or rust was detected on Tasmanian blueberry leaves, stems and fruits during the survey. Blueberry diseases encountered across the farms visited during the surveys were divided into five different groups for ease of description. These were foliar disease, twig tip death or dieback, stem canker, plant death and fruit mould. I: Foliar disease No symptoms of anthracnose or rust were observed on leaves during the field surveys. Subsequently no Colletotrichum species were recovered from the leaf specimens during culturing tests at the laboratory. During the first survey, leaf tip death associated with Alternaria tenuissima (Kunze) Wiltshire was encountered at a farm near Deloraine on 2 year-old potted plants of the variety Elliot and Blue Crop, with less than 5% seedlings showing the symptom for each variety (Figs 1-4, Plate 2). The disease was encountered only once. Relocation of the nursery meant that this was not sampled again during the second survey as the seedlings were no longer available for examination. A leaf spot caused by a Mycosphaerella species (and its anamorph Cercosporidium sp.) was also encountered during the first survey in a 25-year-old orchard at Turners Marsh (Figs 5-8, Plate 2). The leaf disease was found only on a few plants of variety Brigitta and was not observed on the young leaves produced on new season spring growth during the second survey. In addition, two other fungal species, Microsphaeropsis sp. (Fig. 9, Plate 2) and a Harknessia species were found to be associated with necrotic lesions on blueberry leaves. The Harknessia species is morphologically similar to H. globosa Sutton and tentatively identified as H. aff. globosa (Fig. 10, Plate 2). II: Twig-tip death or dieback
A twig-tip death or dieback, also called “winter chill” by local growers, was encountered frequently in almost all the farms we visited during both surveys (Figs 1-9, Plate 3 & Figs 1-3, Plate 4). A number of fungal species was isolated from the twig samples showing dieback symptoms. They were Alternaria alternata, Botrytis cinerea, Botrysphaeria sp., Cladosporium sp., Coniothyrium sp., Cytospora sp (and Valsa sp.), Harknessia sp., Pestalotiopsis spp., Seimatosporium arbuti,
Seimatosporium sp. (aff. S. hakeae), Seimatosporium vaccinii and Sordaria sp. Of these fungal species, Pestalotiopsis spp were the most common species encountered during the surveys. They were found from several farms. Three morphologically different species were identified: Pestalotiopsis sp.1, a species with the majority of conidia having 3 apical appendages and growing fast on agar; Pestalotiopsis sp. 2, a slow growing species with the majority of conidia having 2 apical appendages (Fig. 6, Plate 3) and Pestalotiopsis sp. 3, having 1-2 apical appendages and differentiating from the other two by its conidia having 2-pimented cells, instead of 3 (Fig. 7, Plate 3). The next most common fungal species isolated from twig samples were A. alternata, B. cinerea, Cladosporium sp., Coniothyrium sp. (Fig. 5, Plate 3) and Seimatosporium arbuti (Fig. 8, Plate 4). Each was encountered at least twice during the survey. For all other species, Botrysphaeria sp., Cytospora sp (and Valsa sp.), Harknessia sp. (Figs 1-3, Plate 4) and Sordaria sp (Figs 4-5, Plate 4), each was found once only. III: Stem canker Stem canker was found to be a minor problem, only encountered in a few farms. Necrotic lesions were occasionally observed on young stems/branches, especially young shoots (Plate 4 & 5). These lesions were usually superficial and fungal fruiting bodies could be observed occasionally on the surfaces. During the first survey, an Ascomycete (with two-celled ascospores) (Figs 6-8, Plate 4) and a Pestalotiopsis species (with short apical appendages and different from those isolated from dieback twigs) (Figs 1-4, Plate 5) were found on the lesion surfaces of cankered stems collected from a farm at Frankford, and during the second survey, Zetiasplozna thuemenii was found to be associated with a basal stem canker of a potted Brigitta seedling imported from Victoria. (Figs 5-7, Plate 5). IV: Plant death
Dead plants were occasionally encountered during the survey, eg. one or two individuals each in some orchards (Fig. 1, Plate 6). The majority of those plants had been dead for a long time. Some dead plants were found with stems or twigs covered with fungal fruiting bodies. One twig on the dead plant from a farm at Lilydale was found carrying fruiting bodies with pinkish to orange coloured spore masses. Morphological examination of conidia and cultures recovered from the conidia indicated that this is a species of Colletotrichum, tentatively identified as C. aff. acutatum (Figs 2-8, Plate 6). The fungus was not found during the second survey when the farm was revisited. The dead plant bearing the fruiting bodies of C. aff. acutatum had been removed by the owner. There are still some dead plants in the block, but no fungal fruiting bodies were observed on the dead stems or twigs. Those plants have been long-dead, probably due to abiotic factors such as drought.
Morphologically, C. aff. acutatum detected on the dead twig in Tasmania most closely resembles the races of C. acutatum detected on fruits from NSW, but is different in cultural features (eg. growth rate and colony colour), formation of appressoria from conidia and hyphae (Plate 7 & 8). For example, the appressoria germinated from conidia in C. aff. acutatum have very short germination tubes (Fig. 7, Plate 6), compared with longer germination tubes produced by C. acutatum (race 1) (Figs 6-7, Plate 7) and C. acutatum (race 2) (Fig. 9, Plate 8), although appressoria with short germination tubes can be found in race 2 (Figs 7-8, Plate 8). The appressoria germinated from hyphae in C. aff. acutatum are long, clavate to irregular in shape (Fig. 8, Plate 6), in contrast to the more rounded and regularly-shaped appressoria for both race 1 (Fig. 8, Plate 7) and race 2 (Fig. 10, Plate 8). V: Fruit mould Fruit samples were taken from 16 of 19 farms surveyed where mature berries were available. Fruits at the harvest season were generally healthy and appeared free of diseases when inspected in the field. Apart from common mould species like Penicillium spp., Aspergillus spp., Mucor spp., Rhizopus stolonifer, Cladosporium spp. and Botrytis cinerea, no anthracnose-causing fungi or other diseases were detected on the berries after incubation at either 4°C or 30°C.
Other diagnostic work on fungi associated with death of cuttings in nurseries Fungi associated with cutting death During the survey, severe cutting death was observed at Nursery 1. Up to 30-40% of the cuttings of local origins in some of the trays maintained in both the glasshouse and shade-house were found dead or dying (Fig. 1, Plate 9). Losses due to cutting death were also reported from Nursery 2 and Nursery 3. A number of mitosporic fungi were observed on or isolated from the samples tested. These included: Alternaria alternata, Cladosporium sp., Camarosporium-like fungus, Epicoccum purpurascents, Pestalotiopsis sp1. and a Colletotrichum species from Nursery 1; Pestalotiopsis sp.1 from Nursery 2 and A. alternata, Coniella-like fungus, Coniothyrium sp., Cryptosporiopsis-like fungus and Graphium sp. (and probably its teleomorph, Ceratocystis sp.) from Nursery 3.
Detection of C. aff. gloeosporioides from cuttings Morphological examination of the Colletotrichum species from Nursery 1 indicated that it is the same fungus as C. aff. gloeosporioides found during the investigation on the death of cuttings submitted to us from the same nursery in 2007 (Figs 1-11, Plate 10). Our studies on morphology indicated that C. aff. gloeosporioides is different from C. gloeosporioides detected on blueberries imported from NSW. The significant differences between C. aff. gloeosporioides from Tasmania and C. gloeosporioides from NSW are as follows:
• Shape and size of conidia The Tasmanian C. aff. gloeosporioides has conidia of variable dimensions: some are cylindrical with the apical end obviously swelling (slightly spatulate) (Fig. 8, Plate 10) and cylindrical, wider toward the apical ends (Fig. 9, Plate 10). Conidia are thin and long measuring between 21.7-28.1 x 4.6-5.6 µm and 16.6-20.4 x 3.6-5.6 µm. C. gloeosporioides from NSW on fruits also has cylindrical conidia, the majority of which are wider toward the basal ends and measured 13.0-21.0 x 4.5-6.1 µm (Fig. 3, Plate 11).
• Shape of appressoria Appressoria germinated from conidia and hyphae in C. aff. gloeosporioides are irregular in shape (protuberate) (Figs 10 & 11, Plate 10), whilst the C. gloeosporioides from NSW produced more regular-shaped appressoria: rounded or ellipsoid (from conidia) to fusiform (from hyphae) (Figs 6-10, Plate 11).
• Presence of teleomorph (Glomerella sp.) On artificial media (PA and PDA), C. aff. gloeosporioides readily produced perithecia, asci and ascospores which are similar to those observed on the surface of the stem (Figs.1-5, Plate 10). No structures of the teliomorph were observed under the same culture conditions for C. gloeosporioides from NSW. C. aff. gloeosporioides was found only from Nursery 1 (Table 4).
Table 4: List of blueberry cutting samples from which Colletotrichum aff. gloeosproioides was isolated from Nursery 1
Sample Variety Description Origin
134-07 Brigitta Submitted by the owner on 10/11/2007 Local NBB-37 Brigitta Collected by Z. Yuan on 19/02/2008 Local 70-08-2 Brigitta Collected by D. Metcalf on 1/8/2008 VIC 70-08-5 Brigitta Collected by D. Metcalf on 1/8/2008 VIC 109-08-1 Brigitta Collected by Z. Yuan on 13/11/2008 VIC 109-08-2 Duke Collected by Z. Yuan on 13/11/2008 VIC
Molecular characterisation of Colletotrichum species Assay 1: Using β-tublin-2 sequence analysis, the Colletotrichum species from Tasmania were able to be distinguished from the species from NSW after comparison of the sequence data of Whitelaw-Weckert et al. (2007):
• Two NSW C. acutatum (race 1 and 2) isolated from imported blueberry fruits in 2007 appear to be the same species, but slightly different to each other. They fit within a C. acutatum group called A9 and then subgroup A9b proposed by Whitelaw-Weckert et al. (2007). Subgroup A9b contains other known C. acutatum isolates from blueberry from Corrindi, NSW.
• C. aff. acutatum from the dead plant in Tasmania is different from the NSW isolates of C. acutatum (race 1 and 2). C. aff. acutatum fits within a C. acutatum group A4, which contains C. acutatum isolates from chilli of Wagga Wagga, NSW and an isolate from tomato of Tasmania.
• C. aff. acutatum is different from C. aff. gloeosporioides isolated from cuttings in Tasmania.
• C. aff. gloeosporioides isolate is different to the NSW C. gloeosporioides isolate from imported blueberry fruits and does not fit with any of the Australian isolates of Colletotrichum studied by Whitelaw-Weckert et al. (2007).
• The isolate of C. lupini from lupins is unrelated to others and fits with other lupin infecting races in group A1.
Assay 2: The sequence data on the region of the β-tublin-2 gene were compared with published sequences of Colletotrichum species. A phylogenetic tree is produced (Fig. 1).
0.1
AJ409289 G. tucumanensis (= C. falcatum)
(Saccharum, India,,T)
AF411752 C. gloeosporioides (Vaccinium, US, V)
AJ314713 C. sp. (Citrus, Portugal, T)
DM 134-07.
AY245020 Colletotrichum kahawae (Coffea, Malawi, N)
DM 119-07-C
CGU14138 Colletotrichum gloeosporioides f. sp. aeschynomene (Aeschynomene virginica, USA, B)
DQ991691 DAR69982 (Tomato, Tasmania, W)
DQ991692 DAR76933 (Chilli, NSW, W)
DM BB-167
DQ991701 DAR28076 (Mango, NSW, W)
DQ991683 DAR76907 (Grape, NSW, W)
DQ991684 DAR76908 (Grape, NSW, W)
AF411749 (Fragaria, USA, V)
AJ314717 C2897 (Fragaria, WA, T)
DM 16-04.
DQ991682 DAR76924 (Lupin, NSW, W)
AJ314719 96A4 (Lupin, WA, T)
DM 119-07-B
DM 119-07-A
DQ991710 DAR76913 (Grape, NSW, W)
DQ991712 DAR76921 (Olive, NSW, W)
DQ991693 DAR72407 (Almond, SA, W)
DQ991688 (Blueberry, NSW, W)
AF411751 (Prunus, US, V)
DQ991687 DAR68512 (Tomato, NSW, W)
DQ991686 DAR24831a (Avocado, NSW, W)
Fig. 1: Maximum likelihood tree based on Beta-tubulin sequences of six isolates and published sequences. Bar repesents 10% nucleotide substitution. Sequence accession number is followed by isolate number (for Australian isolates) and in brackets, host, country (or state, if Australian) and a code* for the reference. * B = Buhr & Dickman, 1994; N = no publication liked to the sequence, submitted by the National Coffee Research Centre of Colombia; T = Talhinhas et al. (2002); V = Vinnere et al., (2002) and W = Whitelaw-Weckert et al. (2007).
The result is more or less a duplication of Assay 1 where only Australian isolates were compared.
• Two C. acutatum isolates from NSW (DM119-07-A and DM119-07-B) are clustered in a group including known C. acutatum isolates from grape and olive of NSW, and different from Tasmanian C. aff. acutatum (DM BB-167).
• Tasmanian C. aff. acutatum (DM BB-167) is grouped with C. acutatum isolates from chilli of NSW and Tomato of Tasmania.
• C. gloeosporioides from NSW (DM119-07-C) and C. aff. gloeosporioides from Tasmania (DM134-07) are different, but are both within the C. gloeosporioides group. Isolate DM119-07-C is close to a form species from Aeschynomene virginica of USA: C. gloeosporioides f. sp. aeschynomene.
• Tasmanian C. aff. gloeosporioides from cuttings (DM134-07) is closely related to a species from coffee, Malawi, named C. kahawae.
• The isolate from lupins (DM16-04) is grouped with other lupin infecting races from WA and NSW.
Assay 3: Sequence data on glyceraldehyde-3-phosphate dehydrogenase (GPD) gene were compared with published sequences of Colletotrichum species. A phylogenetic tree is produced (Fig. 2).
0.1
DM 134-07
DQ792849 C. gloeosporioides (L)
COGGPD C. gloeosporioides (T)
DM 119-07C
EU647323 (M) leatherleaf fern USA
DM 16-04
EU647319 (Mc) sweet orange USA
EF593347 (Mr) Orthezia praelonga, Brazil
DM BB-167
DM 119-07A
DM 119-07B
C. acutatum
Fig. 2: Maximum likelihood tree based on GPD sequences of six isolates and published sequences. Bar repesents 10% nucleotide substitution. A code* in brackets for the reference. *L = Liu et al. (2007); Mc = MacKenzie et al. (2007); Mr = Marcelino et al. (2008) and T = Templeton et al. (1992).
The result from above analysis using GPD gene sequences is similar to the results obtained in assay 1 and 2 using Beta-tublin gene sequences.
• Six isolates are divided into three groups: four in C. acutatum group (DM119-07-A, DM119-07-B, DM BB-167 and DM16-04), one in C. gloeosporioides group (DM119-07-C) and the third group represented by a single isolate (DM134-07).
• Within the C. acutatum group, Tasmanian C. aff. acutatum (DM BB-167) is separated from two C. acutatum isolates (DM119-07-A & B) from NSW by 20% nucleotide substitutions.
• Isolate DM119-07-C from NSW fits well with other two C. gloeosporioides within that group.
• The single-isolate group of Tasmania, C. aff. gloeosporioides (DM134-07) is isolated from other two groups, indicating it is different species and not a C. gloeosporioides, which supports the result of assay 2.
DISCUSSION Blueberry Rust Pucciniastrum vaccinii usually produces spots on leaves or causes leaf death, if the disease is severe (Caruso and Ramsdell, 1995). The detection of this rust disease on fruits imported from NSW appears unusual. Pustules or uredinia formed by P. vaccinii are rarely seen on fruits. In most publications on this rust disease, no information on infected fruits is given, although symptoms on leaves are always fully described. There are 39 collections of the rust recorded on blueberry in APPD from NSW, all on leaves (APPD, 2009). A literature search has turned out only one report that briefly describes the disease on fruits as "as much as 85% of the fruits on symptomatic bushes showed pustules on the scar area." (Barrau et al., 2002). This description fits well with the symptoms observed on the blueberry fruits from NSW, from which a high infection incidence of 5.4% (n=466) was detected (Yuan, 2007, unpublished data). P. vaccinii is a heteroecious species with uredinia and telia on blueberries and aecia on the alternate host: hemlock, but can cycle on blueberries in the absence of the alternate host (Caruso and Ramsdell, 1995). There are no records of any rust on hemlock in Australia (APPD, 2009). Apart from Vaccinium spp., this fungal species also has been recorded on host plants like Azalea spp., Gaylussacia baccata, Lyonia ligustrina, Menziesia ferruginea, Oxycoccus spp., Pieris villosa, Rhododendron spp. and Tsuga sp. (Farr et al., 2009). During the survey, the rust disease was not detected on any aboveground parts including fruits. It has been listed as a “List A disease” for Tasmania (DPIW, 2008). The negative result from the present surveys supports the continued application of import restrictions upon host material, including blueberries. Blueberry Anthracnose Blueberry anthracnose occurs in many parts of the world. Two species of Colletotrichum: C. acutatum and C. gloeosporioides are reported to cause this disease (Caruso & Ramsdell, 1995; Daykin & Milholland, 1984; Verma et al., 2006). Both species have been recorded on blueberry in Australia. C. acutatum is recorded in New South Wales and Victoria, and C. gloeosporioides in New South Wales and Western Australia (APPD, 2009). There are no official records of any Colletotrichum species on blueberry in Tasmania prior to the present surveys. During the surveys, no anthracnose was detected on leaves and fruits, but two Colletotrichum species were detected respectively on a stem of a dead plant in an orchard and on cuttings in a nursery. The species from the dead plant was tentatively identified as C. aff. acutatum and the one from cuttings as C. aff. gloeosporioides. Our studies in morphology and DNA sequence analyses using β-tublin-2 gene and GPD gene have indicated that both species from Tasmania are different
from all three Colletotrichum species (races) detected on blueberry fruits imported from New South Wales. In a study on phylogenetic relationships and pathogenicity of Colletotrichum acutatum isolates from grape in subtropical Australia, Whitelaw-Weckert et al. (2007) clustered 63 Australian C. acutatum isolates from wine grapes and other horticultural crops into six groups, namely A1, A2, A3, A4, A5 and A9 (with subgroups A9a and A9b). When compared with the published DNA sequences for the C. acutatum groups from Australia (Whitelaw-Weckert et al., 2007) and from overseas (Buhr & Dickman, 1994; Talhinhas et al., 2002; Vinnere et al., 2002), the β-tublin-2 gene sequences for our C. aff. acutatum (DM BB-167) were fitted into the C. acutatum group A4, along with isolates from chilli collected from Wagga Wagga, NSW and from tomato collected from Tasmania. (Assay 1 & 2), suggesting that the C. aff. acutatum race from Tasmania is a different race of C. acutatum to the one from NSW. The DNA analysis using GPD sequences also confirmed that C. aff. acutatum from Tasmania is a race of C. acutatum, but which is genetically different from the two races of C. acutatum of NSW (Assay 3). The isolates from both chilli and tomato (Fig. 1) have been tested for their pathogenicity on blueberry and the visible evidence of disease at 14 days after inoculation was scored “0” (as <30% of berries were infected). However, all five C. acutatum isolates clustered in subgroup A9b from blueberry of Corrindi and Coff’s Harbour, NSW are highly pathogenic to blueberry, grape and strawberry, with visible evidence of disease at 14 days after inoculation, and these all scored “++” (≥ 60% infected) on blueberry, grape, and strawberry (three isolates) (Whitelaw-Weckert et al., 2007). The close genetic relationship between the Tasmanian C. acutatum collected from tomato and C. aff. acutatum collected from blueberry may be of significance with respect to host range of the isolate. If they are the same species, the evidence so far could suggest that C. aff. acutatum is not a host-specific fungus and is only weakly pathogenic to blueberry. The fact that we encountered it only once on a dead blueberry twig suggests that it is not specifically adapted to infection of blueberry, and that it has not established as a pathogen on blueberry plants in Tasmania. Using the same β-tublin-2 gene sequence data as for C. aff. acutatum, it was found that the Tasmanian C. aff. gloeosporioides isolated from blueberry cuttings in the nursery did not match C. gloeosporioides or any other known Colletotrichum species, when compared only with Australian species in Assay 1. However, when the comparison was enhanced with non-Australian species included, the Tasmanian C. aff. gloeosporioides from cuttings (DM134-07) is separated from C. gloeosporioides of NSW (DM119-07-C), but closely clustered with a species from coffee, Malawi, named C. kahawae (Assay 2). The analysis using GPD sequences also reveals that C. aff. gloeosporioides is not conspecific to C. gloeosporioidies, as demonstrated by the maximum likelihood tree that separates it from three C. gloeosporioides isolates, including the isolate from NSW fruits (DM119-07-C) (Assay 3).
The close genetic relationship between C. aff. gloeosporioides and C. kahawae is interesting, given the vast differences between these two fungi in host, geographic distribution and the pathogenic status. C. kahawae, a name proposed recently, is a well know pathogen of coffee berries with records dating back to 1926 under the synonym C. coffeanum or C. coffeanum var. virulans (Waller et al., 1993). This fungus has not been recorded in Australia (APPD 2009). Further work on the identification of this organism is required to confirm its status. Morphological taxonomic features of the two species are quite close, but DM134-07 is probably closer to C. gloeosporioides. C. aff. gloeosporioides has been encountered a few times on blueberry cuttings, but only in one nursery since its first discovery on a cutting in 2007. Cutting death appeared to be a severe problem in that nursery. Laboratory culturing tests of the cutting and seedling samples from that nursery yielded a number of fungal species. It is difficult to establish a direct connection between the cutting death occurring in the nursery with a particular fungal pathogen, including this Colletotrichum species. Cutting death was also reported from other nurseries. Samples of dead cuttings were collected from these nurseries and no Colletotrichum species were recovered from these samples. The fact that C. aff. gloeosporioides was found on a few occasions from a single location (Nursery 1), regardless of the origin of the cuttings investigated, may indicate it is localised in that particular nursery. The pathogenicity of C. aff. gloeosporioides was not tested during the survey and its pathogenic status remains unknown. However, other evidence indicated that fungus may be a weak or secondary pathogen of blueberry. For instance, although the fungus was isolated from dead cuttings a few times, it was never a predominant fungal species recovered during each incidence. In addition, follow-up surveys for anthracnose on the plants released from that nursery were negative. Trace back to local properties from which cuttings were sourced was also negative. Following examination of 3332 plants (a minimum number of 3253 required) and 16,284 berries (a minimum of 12,500 required), we are 95% confident that blueberry anthracnose occurring in mainland Australia is not present in berries and plants in Tasmania at a prevalence of 0.1% or higher. In our surveys, we employed a symptom-based sampling method with which only plant tissues showing disease symptoms were examined, and there are possibilities that we failed to detect latent anthracnose during our sampling process. However, the probability of detection by this method has been weighted by taking into account a test deficiency of 10% for false negatives, including latent infections. Our morphological and molecular studies on the original isolate of C. lupini (DM 16-04) ) from lupins during the incursion in Tasmania (Figs 1-7, Plate 12) have also demonstrated that it is unrelated to all Colletotrichum species on blueberry and other hosts and appears to be host-specific to lupins as those lupin races from WA and NSW. Lupin anthracnose caused by C. lupini is not established, and is a List A disease for Tasmania and subject to Import Requirements (DPIW, 2008).
Other Blueberry Diseases There were no systematic surveys on blueberry diseases in Tasmania, prior to the current surveys. Only one bacterial disease, Crown Gall caused by Agrobacterium tumefaciens, was previously recorded on blueberry in Tasmania and no disease associated with fungi was officially reported (APPD, 2009). More than 20 fungal species were encountered on leaves, stems, twigs, fruits and cuttings during the current survey, where the emphasis was on surveying anthracnose on above-ground plant parts. Root diseases were not attended. These fungal species were found to be associated with five groups of blueberry diseases: foliar disease, twig tip death or dieback, stem canker, plant death and fruit mould. However, none of these diseases appeared to be having significant plant health impacts. The fungi encountered, the type of disease caused and their status in Australia and Tasmania are summarised in Table 5 and discussed in more detail below. Table 5: Summary of fungal pathogens encountered during the survey
Fungi Incidence Disease type Previously recorded on blueberry (A) or other plants (B) in Australia Tasmania (A/B) (A/B)
Alternaria alternata +* Twig dieback yes/yes no/yes & cutting death Alternaria tenuissima + Leaf spot no/yes no/no Aspergillus spp. +++ Storage fruit rot no/yes no/yes Botryosphaeria sp. + Twig dieback yes/yes no/yes Botrytis cinerea ++ Storage fruit rot yes/yes no/yes & twig dieback Cercosporidium sp. + Leaf spot no/yes no/yes Cladosporium spp. +++ Storage fruit rot no/yes no/yes Twig dieback Colletotrichum aff. acutatum + Dead twig yes/yes no/?** C. aff. gloeosporioides + Cutting death no/no no/no*** Coniothyrium sp. ++ Twig dieback no/yes no/yes Cytospora sp. + Twig dieback yes/yes no/yes Epicoccum purpurascents ++ Twig dieback no/yes no/yes & cutting death Harknessia aff. globosa + Leaf lesion no/no no/no Harknessia sp. + Twig dieback no/? no/? Mucor spp. +++ Storage fruit rot no/yes no/yes Mycosphaeriella sp. + Leaf spot yes/yes no/yes Microsphaeropsis sp. + Leaf lesion yes/yes no/yes Penicillium spp. +++ Storage fruit rot no/yes no/yes Pestalotiopsis spp. ++ Twig dieback, yes/yes no/yes stem canker & cutting death Rhizopus stolonifer +++ Storage fruit rot no/yes no/yes Seimatosporium arbuti, ++ Twig dieback no/yes no/no Seimatosporium aff. hakeae + Twig dieback no/yes no/yes Seimatosporium vaccinii + Twig dieback no/no no/no Sordaria sp. + Twig dieback no/yes no/no Valsa sp. + Twig dieback no/yes no/yes Zetiasplozna thuemenii + Stem canker no/no no/no
*+ = minor, encountered only once or twice at one property or not the dominant organism isolated for the disease type; ++ = moderate, encountered at 2-10 properties; +++ = common, encountered at nearly all properties.
**? = status in Australia or Tasmania unsure, due to the uncertainty of species identification. ***Bold faced = new record for Australia.
Foliar diseases were encountered very rarely during the surveys. Among the fungal species examined on leaves, only two, Alternaria tenuissima and Mycosphaeriella sp. were found to be associated with well-developed leaf spots and could be primary pathogens. Alternaria tenuissima is a common and widespread species and often found on dead plants. It has been recorded as a leaf spot pathogen of blueberry in USA (Farr et al., 2009). In Australia, this fungus is recorded causing fruit rot, leaf spot, seed mould and stem lesions in Victoria, New South Wales, Western Australia, Queensland and South Australia on a range of plants (APPD, 2009). Prior to the present survey, A. tenuissima has not been recorded on blueberry in Australia. Blueberry leaf spot caused by other Alternaria species, eg. A. alternata has been recorded from NSW and VIC. Mycosphaerella leaf spot is rarely recorded on blueberry crops worldwide, although there are 4 records from NSW in Australia (APPD, 2008). This leaf spot found at Turners Marsh is the first record on blueberry crop in Tasmania. Mycosphaerella species are generally considered host-specific and can have different anamorphic stages belonging to hyphomycetes or coelomycetes (Kiffer & Morelet, 2000). The anamorph, Cercosporidium sp., has not been recorded on blueberry, but species of Cercosporidium have been reported on a number of other plants in Tasmania and mainland Australia (APPD, 2008). The species of Microsphaeropsis is morphologically close to M. callista (H. Syd.) Sutton, having slightly smaller and narrower, fusiform conidia measured 5.5-7.0 x 2.5-4.5 µm than those of M. callista on eucalypts (measured 7-8.5 x 4.5-5.5 µm) (Sutton, 1980). M. callista is recorded on eucalypt leaves and litter in NSW, QLD and Tasmania (Yuan, 1999). Harknessi globosa described on dead Eucalyptus globulus from New Zealand has not been recorded in Australia (APPD, 2008). The fungus found on blueberry leaves fits with the description for H. globosa given by Sutton (1980) and tentatively identified as H. aff. globosa. About 10 species of Harknessia have been recorded in Australia, mainly on eucalypts. This is the first time that a Harknessia species is found on a species in the genus Vaccinium. Farr et al. (2009) have recorded the genus Harknessia on a wide range of host plants, but not on Vaccinium. Twig dieback or “winter chill” was the most commonly encountered disease across all the blueberry farms inspected. It appears to be the most significant disease/physiological issue faced by growers at the present. The cause of the phenomenon is unknown and could be variable with orchards. A number of fungal species were examined or isolated from the dying or dead twig samples. The majority of twigs showing the symptoms were found having signs of old mechanical wounds (eg. from pruning or harvesting), suggesting a wound-related secondary infection. Most fungal species isolated could probably be secondary pathogens.
Outside Australia, blueberry twig dieback has been recorded frequently in commercial plantings in Chile (Espinoza et al., 2008). Two Pestalotiopsis species (P. clavispora and P. neglecta) and Truncatella angustata (a fungus morphologically close to Pestalotiopsis having conidia of two pigmented cells like P. sp 3 found in our survey) are reported to be associated with the dieback in Chile and have been proved to be primary pathogens for blueberry dieback and cankers by inoculation tests. This appears to be in common with our current survey results. Several Pestalotiopsis species were encountered from stem, twig and cutting samples during the survey. They were the most common fungal species isolated from dying twigs. Pestalotiopsis sp.2, for instance, was predominantly recovered from all five samples tested from one farm with a percentage of isolation ranging from 9-50%. Further studies on morphology and pathogenicity of these Pestalotiopsis fungi are needed. Apart from Pestalotiopsis spp., Coniothyrium sp. found twice during the survey can be a potential candidate as a primary pathogen. Some species of Coniothyrium are highly pathogenic and can cause severe plant diseases. The stem canker of eucalypts caused by C. zuluense, for example, is one of the most serious diseases affecting eucalypt plantation development in South Africa (Old et al., 2003). In Tasmania, stem cankers associated with Coniothyrium species have been recorded on several horticultural plants, such as peony, rose, raspberry and loganberry (Sampson & Walker, 1982). Three Seimatosporium species, S. arbuti, S. aff. hakeae and S. vaccinii were isolated from dieback twigs during the survey. Only one unnamed species of Seimatosporium has previously been recorded on blueberry stems from NSW in Australia (APPD, 2009). S. arbuti is recorded on leaves of Cydonia oblonga and Acacia pycnantha in NSW and VIC respectively (APPD, 2009). S. hakeae has also been recorded in NSW, VIC, WA and TAS on leaves of Hakea spp. and Grevillea sp. (APPD, 2009). Both S. arbuti and S. aff. hakeae are, for the first time, recorded on blueberries. S. vaccinii has not been previously recorded in Australia, but on stems of Vaccinium spp. in New Zealand and Switzerland, on Staphylea trifolia in USA and Crataegus oxyacantha in UK (Sutton, 1980). The pathogenicity of these species remains unknown, although two species of Seimatosporium, S. lichenicola and S. rhodendri are reported to cause cankers of blueberries in USA (Farr et al, 1989). A Botryosphaeria species was for the first time recorded on blueberry in Tasmania. Two Botryosphaeria species have been reported causing blueberry stem cankers or stem blight in USA (Caruso & Ramsdell, 1995). They are B. corticis (Demaree & Wilcox) Arx & Müller and B. dothidea (Moug.:Fr.) Ces. & De Not. None of these two species are recorded on blueberry in Australia, although three other Botryosphaeria species: B. australia, B. ribis and Botryosphaeria sp. are present in NSW and Victoria (APPD, 2009). Many species in the genus Botryosphaeria are woody plant pathogens. All other fungal species isolated from blueberry twigs are less likely to be responsible for the dieback. Alternaria alternata is an extremely common and cosmopolitan species occurring on many kinds of plants. It is mainly
saprophytic, but can cause plant diseases like fruit rot, leaf spot and seed rot (Ellis 1971). Botrytis cinerea is a cosmopolitan species causing “grey mould” of flowers, leaves, stems and fruits of all sorts of plants (Ellis 1971). It can be pathogenic under certain circumstance (like glasshouses). Species of the genus Cladosporium are generally saprophytic or occur as a secondary invader on many different plants (Ellis 1971). Cytospora species are common on woody plants causing stem cankers, and can be weak pathogens or endophytes (Adams et al., 2006). Epicoccum purpurascents is an extremely common secondary invader on all sorts of plants. It is cosmopolitan and has been isolated from soil, air and textiles (Ellis, 1971). Harknessia sp. examined on a potted seedling showing twig dieback symptoms is the first species of Harknessia found on blueberry twigs, apart from H. globosa on leaves. This fungus most closely resembles H. ventricosea Sutton & Hodges, a species described on dead eucalypt leaves from USA, in conidia shape and size (Sutton, 1980). H. ventricosea is only reported to be associated with leaf spots. Species of the genus Sordaria are generally considered as saprophytes on dung and occasionally on plants (seeds or wood) (Hanlin, 1990). Stem cankers were only encountered in a few farms during the survey. Canker lesions were usually superficial and fungal fruiting bodies could be observed occasionally on the surfaces. Only a few fungal species were observed. Among these, Zetiasplozna thuemenii, a fungus with conidia bearing distinctively characterised appendages is worth mentioning. The fungus was found at the basal stem canker of a potted Brigitta seedling imported from Victoria. No previous species of Zetiasplozna were recorded in Australia. Z. thuemenii has been described only from Italy, Mexico, Ukraine and USA on leaves and fruits of Metasequoia, Pistacia, Psidium and Vitis (Nag Raj, 1993). The pathogenic status of this species remains unknown. Dead mature plants were occasionally observed in some farms during the survey. They can be potential inoculum sources for new diseases, if bearing fungal fruiting bodies. Removal and destruction of these dead plants are good disease management practices. C. aff. acutatum detected on the stem of one dead blueberry plant during the first survey was not found during the second survey when the farm was revisited. The dead plant bearing the fruiting bodies of C. aff. acutatum had been removed by the owner. Not many important fruit diseases, apart from anthracnose, have been reported for blueberry, either in the field or in storage, worldwide. In USA, Botryosphaeria vaccinii (Shear) Barr, the causal agent of a blueberry leaf spot and berry speckle can also cause fruit dry rot during the green stage or pre-ripening period and Phomopsis vaccinii Shear is also reported causing soft, often split, fruits (Caruso & Ramsdell, 1995). In this survey, no diseases were recorded on fruits, but some common moulds were observed when the fruits were incubated under either low or high temperature (4 & 30°C). Fruit rot caused by common mould fungi, such as Aspergillus spp., Penicillium spp. and Cladosporium spp. in storage have been documented for cranberry in USA (Caruso & Ramsdell, 1995).
Although the pathogens causing blueberry anthracnose, C. acutatum and C. gloeosporioides, can attack the aboveground parts of plants and develop various symptoms on leaves, twigs and stems, they primarily affect fruits causing fruit rot (Caruso & Ramsdell, 1995). The anthracnose fruit rot is a serious and major problem reducing yield and postharvest fruit quality worldwide. Crop loss up to 20% has been reported in USA (Caruso & Ramsdell, 1995). There are no official reports of crop loss due to anthracnose in Australia. During the routine inspection in 2007, Quarantine Tasmania intercepted blueberries from NSW with an incidence of anthracnose as high as 5.8% (n=466) (Yuan, 2007, unpublished data). The high infection of the fruits from NSW by anthracnose was thought due to the time from harvest to market in Tasmania and the fumigation treatment required by Tasmanian quarantine regulations (S. Kumar 2007, personal communication), as during the process, berries have to be warmed up to room temperature and this predisposes it to post-harvest rots. However, studies on blueberry anthracnose have revealed that it is a pre-harvest disease, although post-harvest rot occurs during storage (Caruso & Ramsdell, 1995; Hartung et al., 1981; Yoshida et al., 2007). Fruit rot usually occurs on berries as they mature and infected fruits can remain symptomless until maturity or after harvest. An inoculation test conducted in USA has shown that inoculation of swelling flower buds, blossoms and immature fruit, both on mature branches of the cv. Jersey bushes in the field, and on potted 3- to 4-yr-old bushes resulted in apparently healthy fruit that later developed a rapid decay after harvest (Hartung et al., 1981). The disease produces prominent rot symptoms on fruits, which can be readily identified by means of a hand lens or under a dissecting microscope. It is unlikely to be missed if an anthracnose is present. During the present survey, up to 16,284 fresh berries, which were also incubated at either 4°C or 30°C, imitating storage conditions, were inspected for anthracnose and none of these was positive, making us highly confident that fruit rot strains of anthracnose are not present in Tasmania. Cutting death Blueberry cutting death appeared to be common across the industry. Apart from the severe cutting death found in Nursery 1, verbal reports from other nurseries (eg. Nursery 2 and 3) also suggest a high mortality rate in blueberry cuttings. Like the twig dieback frequently observed in Tasmanian orchards, a number of fungal species have been recovered from dying or dead cuttings. However, there is no direct evidence suggesting a single predominant causal agent. The fungi associated with cutting death varied with the nurseries investigated. For example, only one fungal species, Pestalotiopsis sp.1, was isolated from cuttings submitted by Nursery 2. This fungus was observed on one of the nine cuttings submitted and was subsequently recovered from all the cuttings (94%), in contrast to Nursery 3 where a number of other fungal species were isolated, each with a low rate of isolation: Alternaria alternata (2.9%), Coniothyrium sp. (10.3%), Coniella-like fungus (3.0%), Graphium sp.
(and Ceratocytis sp.) (5.9%) and Cryptosporiopsis-like species (observed on one cutting). No species of Pestalotiopsis was detected from this nursery. Similarly a variety of fungal species were isolated from cuttings from Nursery 1: A. alternata (12%), E. purpusascents (18%), Pestalotiopsis sp.1 (38%), Cladosporium sp. (3%), Camarosporium-like fungus (observed on one cutting) and C. aff. gloeosporioides (7%), in addition to Cylindrocarpon sp. (77%) in the first incidence in 2007.
CONCLUSION Colletotrichum acutatum and C. gloeosporioides races occurring in mainland Australia have not been detected in Tasmania. Two different species, C. aff. acutatum and C. aff. gloeosporioides were encountered during the survey, with the former species found on a single dead twig and the later frequently from a single location from which it does not appear to be spreading. Our morphological and molecular studies have revealed that two Colletotrichum species encountered on the dead plant and cuttings during the survey are neither the C. acutatum or C. gloeosporioides race present in NSW. The results from this investigation support our view that there are (virulent) races of C. gloeosporioides and C. acutatum in NSW that are not in Tasmania, and that it would also appear that there may be races in Tasmania that are not in NSW, but which are non-virulent, and very rare in Tasmania. It was determined that:
• The Tasmanian C. aff. acutatum is genetically different from C. acutatum (race 1 & 2) of NSW.
• C. aff. acutatum is genetically related or probably conspecific to the isolate of C. acutatum from tomato in Tasmania. C. aff. acutatum is a non host-specific, saprophytic species, which has not established as a pathogen on blueberry in Tasmania.
• C. gloeosporioides from fruits of NSW is a true C. gloeosporioides. The Tasmanian C. aff. gloeosporioides is morphologically and genetically different from C. gloeosporioides from NSW.
• C. aff. gloeosporioides is not a true C. gloeosporioides and possibly conspecific to C. kahawae. C. aff. gloeosporioides is tentatively identified as C. aff. kahawae, but is referred to in the report as C. aff. gloeosporioides.
• C. aff. gloeosporioides could be a weak pathogen on blueberry but has not been encountered outside the nursery where it was recovered on four occasions. Trace back and trace forward investigations failed to find any incidence of C. aff. gloeosporioides.
• No anthracnose was found on fruits during inspection of 16,284 fruits, either freshly harvested or after incubation at different temperatures. This is in contrast to the high incidence of anthracnose fruit rot in the fruits from NSW detected in 2007.
• The original isolate of C. lupini from lupins in Tasmania during the incursion in 2004 is unrelated to all Colletotrichum species on blueberry and other hosts and appears to be as host-specific to lupins as those from WA and NSW.
On the above basis, it is concluded that Tasmania is free from C. acutatum and C. gloesporioides races causing anthracnose. This survey has shown that no severe disease problems are present on leaves, stems and fruits in commercial blueberry orchards in Tasmania. Twig dieback or “winter chill” was the most commonly encountered symptom and a wide range of fungal species was isolated from dieback twigs. Although several of the fungi obtained in this survey are known to be pathogenic or belong to genera which contain recognised pathogens, e.g. Botryosphaeria sp., Coniothyrium sp., Pestalotiopsis spp. and Seimatosporium spp., except for Pestalotiopsis spp., they were only encountered occasionally and impact of damage is currently minor. Our preliminary morphological studies have shown a number of fungi encountered during the survey are new records for Australia or new to Tasmania. Further taxonomic studies on these fungal species by other fungal taxonomic experts are needed to confirm the identifications. As well as demonstrating freedom from blueberry anthracnose, this survey has provided baseline information that will be invaluable for future fungal diagnosis and assessments of potential impact. Identification of more than 20 fungal species on blueberry for Tasmania during the relatively short period of the survey indicated how little is known about fungi on blueberry in Tasmania and possibly Australia, and their potential plant health significance.
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Plate 1: Blueberry rust and anthracnose intercepted from fruits imported from NSW
Figs 1-4: Rust caused by Pucciniastrum vaccinii, symptoms on fruits (1), close-up view of uredina (2 & 3) and urediniospores (4); Figs 5-8: Anthracnose caused by Colletotrichum acutatum and C. gloeosporioides, symptoms on fruits (5) and close-up view of conidia masses of C. acutatum (race 1) (6), C. acutatum (race 2) (7) and C. gloeosporioides (8). (Bar = 10 µm)
1 2 3
4
5
6
7
8
Plate 2: Foliar Disease
Figs 1-4: leaf spot associated with Alternaria tenuissima (conidiophores and conidia); Figs 5-8: leaf
spot caused by Mycosphaerella sp (ascospores, longitudinal section of an ascoma) and its
anamorph Cercosporidium sp. (conidiophores and conidia); Fig. 9: Microsphaeropsis sp.(conidia);
Fig. 10: Harknessia aff. globosa (conidia). (Bars = 20 µm)
1
2 3 4
5
6
7 8
9 10
Plate 3: Twig Tip Death
Figs1-4: Symptoms of shoot blight (and internal discolouration, inserted photo); Fig. 5:
Coniothyrium sp. (conidia); Fig. 6: Pestalotiopsis sp.2 (conidia); Fig. 7: Pestalotiopsis sp. 3
(conidia); Fig. 8: Seimatosporium arbuti (conidia). (Bars = 10 µm)
1 2
3 4 5
6
7 8
Plate 4: Twig Tip Death and Stem Canker
Figs 1-3: Seedling showing twig tip death associated with Harknessia sp. (symptom, close-up view of conidiomata and conidia); Figs 4 & 5: Sordaria sp. (perithecia on twigs and ascospores); Figs 6-8: Ascomycete observed on a stem canker (canker lesion on stem, ascospores and longitudinal section of an ascoma). (Bars = 20 µm)
1 3
2
7
4
5 6 8
Plate 5: Stem Canker
Figs 1-4: Stem cankers associated with Pestalotiopsis sp. (fruiting bodies, conidia and longitudinal section of a conidioma); Figs 5-7: Seedling with a basal stem canker associated with Zetiasplozna thuemenii (symptom, close-up view of conidiomyata and conidia). (Bars = 20 µm)
7 5
6
1
2
3
4
Plate 6: Colletotrichum aff. acutatum isolated from a dead plant in Tasmania
Fig.1: Dead blueberry plants (Blue Rose, Block 3); Figs 2-3: Colletotrichum aff. acutatum (fruiting bodies and longitudinal section of conidioma); Fig. 4: Conidia of C. aff. acutatum; Figs 5-6: 7-day-old colonies of C. aff. acutatum (top and reverse view); Fig. 7: Appressoria from conidia (24 hours after germination); Fig. 8: Appressoria from hyphae. (Bars = 20 µm)
1 2
3
4 5
6
7 8
Plate 7: Colletotrichum acutatum (race 1) isolated from blueberry fruits from NSW
Anthracnose on fruits (orange spore masses)
Conidia
4-d-old colony on PDA (top view)
4-d-old colony on PDA (reverse view)
Appressoria from conidia
Appressoria from conidia
Appressoria from hyphae (Bars = 10 µm for all the figs
scaled)
1
2
3
4
5
6
7
8
Plate 8: Colletotrichum acutatum (race 2) isolated from blueberry fruits from NSW
Anthracnose on fruits (apricot spore masses)
Longitudinal sections of acervuli
Conidia
4-d-old colony on PDA (top view)
4-d-old colony (reverse view) Appressoria from conidia (laterally)
Appressoria from hyphae (Bars = 10 µm for all the figs scaled)
1
2
3
4
5
6
7 8
9
10
Plate 9: Cutting Death
Fig. 1: Cutting death in trays; Figs 2-3: Dead cuttings bearing fruiting bodies (orange-coloured
conidia masses) of Colletotrichum aff. gloeosporioides; Fig. 4: Dead cutting bearing fruiting bodies
of Epicoccum purpurascents (black spore masses); Fig. 5: Symptoms of cutting death at different
stages of development; Figs 6-7: Pestalotiopsis sp.1 (fruiting bodies and culture colonies on agar);
Fig. 8: Culture colonies of E. purpurascents (reverse view)
1 2 3
4 5
6
7 8
Plate 10: Glomerella sp. and Colletotrichum aff. gloeosporioides on blueberry cuttings
Ascospores Ascus Perithecia
Conidia (slightly spatulate)
Conidia (typically cylindrical)
5-d-old colony on PDA (top view)
Appressoria from conidia (Bars = 10 µm for all the figs scaled)
Appressoria from hyphae
5-d-old colony on PDA (reverse view)
1
2
3 4 5
6
7
8
9
10 11
Plate 11: Colletotrichum gloeosporioides isolated from blueberry fruits from NSW
4-d-old colony on PDA (top view)
4-d-old colony on PDA (reverse view)
Appressoria from hyphae (Bars =10 µm for all the figs scaled)
Appressoria from conidia
Conidia Anthracnose on fruits (dark spore masses)
1
2
3
4
5
6
7
8
9
Plate 12: Colletotrichum gloeosporioides (=C. lupini) isolated from lupins
Fig.1: Conidia; Fig.2: Appressoria from conidia (after 24-hours of incubation); Figs 3-4: 7-day-old culture colony (top and reverse view); Figs 5-6: 12-day-old culture colony (top and reverse view): Fig.7: Appressoria from hyphae. (Bars = 10 µm)
1
2
3
4
5
6 7