First Report of Apple Bitter Rot Caused by Colletotrichum godetiae in the United Kingdom

Apple is an important crop in United Kingdom, with a total production of 233,750 tonnes in 2011. Symptoms of apple bitter rot were observed on apple fruits (Malus domestica L.) in the Newcastle area, United Kingdom, in October 2008. Lesions were round, 1 to 5 cm in diameter, brown and dry, with acervuli producing yellowish spore masses in concentric bands. Infected material was sent to the W-HRI (University of Warwick) for identification of the causal agent. Fungal isolates with morphological characteristics similar to those of Colletotrichum acutatum sensu lato were isolated from diseased fruits. Monoconidial isolates were grown on PDA at 25°C with a 12-h light period. The cultures were light gray, with cottony aerial mycelium getting darker with age and with color ranging from whitish to dark gray on the reverse side of the colony. The cultures have yellowish spores masses and dark melanized structures similar to acervuli. Colletotrichum spp. are difficult to identify solely on morphology; therefore, representative isolates were used for multi-locus gene sequencing and characterization (1). Genomic DNA was extracted using a modified Chelex100 protocol. Three loci were amplified and sequenced: the ITS region was amplified and sequenced using the universal primers ITS4 and ITS5. Primers TB5 and TB6 were used for the amplification and sequencing of the variable region of the TUB gene. Primers GDF1 and GDR1 were used to amplify a 200-bp intron region of the GAPDH gene. No differences were found among the strains at any of the loci. One sequence for each locus has been deposited in GenBank under accessions KF834206 (ITS), KF834207 (TUB), and KF834208 (GAPDH). In GenBank, ITS sequences matched with 100% identity to C. higginsianum (EU400147) and to C. gloeosporioides (AJ301931 to 972); and with identity between 99.6 and 99.8% with sequences belonging to C. godetiae (part of C. acutatum species complex). The TUB sequences match with 100% identity to more than 25 sequences belonging to C. godetiae. The GAPDH sequences match with 100% identity to JQ948739 and 35 belonging to C. godetiae strains IMI 381927 and CBS 131331. A multilocus phylogenetic tree (ITS, TUB, and GAPDH) was reconstructed using sequences of reference strains belonging to C. higginsianum, C. gloeosporioides, C. godetiae, and related species. The phylogenetic tree confirmed the identity of the strains isolated from apple as C. godetiae. Koch's postulates were tested with representative isolate by artificial inoculation of 12 healthy fruits of the cv. Golden Delicious. Fruit surfaces were sterilized with 70% ethanol, wounded with a sterile needle, and then inoculated with a plug of actively growing mycelium prepared from a 10-day-old culture grown on PDA. Inoculated fruits were incubated in sterile conditions at 25°C with a 12-h photoperiod. In 83% of fruits, symptoms appeared between 7 and 15 days later. The rot begins as light brown, circular lesion getting darker with orange spore masses. Fungal colonies isolated from the lesions and cultured on PDA have identical morphological characteristics of the isolate used for the pathogenicity assay. To the best of our knowledge, this is the first report of apple bitter rot caused by C. godetiae in the United Kingdom. Apple bitter rot is spread worldwide and in moist, temperate regions it is considered one of the most important diseases causing considerable crop losses. Since the losses are more severe under prolonged warm and wet weather conditions, bitter rot caused by C. acutatum species may become an emerging problem in the United Kingdom in the near future, and may require investigation of management practices to control this new disease. References: (1) R. Baroncelli. Colletotrichum acutatum sensu lato: From diversity study to genome analysis. Coventry, United Kingdom, PhD thesis, 2012. (2) U. Damm et al. Stud. Mycol. 73:37, 2012.

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First report of preharvest fruit rot of ‘Pink Lady’ apples caused by Colletotrichum fructicola in Italy

Plant Disease ◽

10.1094/pdis-11-20-2404-pdn ◽

2021 ◽

Author(s):

Marcel Wenneker ◽

Khanh Pham ◽

Engelien Kerkhof ◽

Dalphy O.C. Harteveld

Keyword(s):

Species Complex ◽

Management Strategies ◽

Morphological Characteristics ◽

Late Summer ◽

Fruit Rot ◽

Colletotrichum Acutatum ◽

Apple Orchards ◽

First Report ◽

Bitter Rot ◽

Colletotrichum Fructicola

In late summer 2019, a severe outbreak of fruit rot was observed in commercial ‘Pink Lady’ apple orchards (>20 ha in total) in the region Emilia-Romagna (Northern Italy). The symptoms on the fruit appeared as small circular red to brown lesions. Disease incidences of over 50% of the fruits were observed. To isolate the causal agent, 15 affected apples were collected and small portions of fruit flesh were excised from the lesion margin and placed on potato dextrose agar (PDA). The plates were incubated at 20°C in the dark, and pure cultures were obtained by transferring hyphal tips on PDA. The cultures showed light to dark gray, cottony mycelium, with the underside of the culture being brownish and becoming black with age. Conidia (n=20) were cylindrical, aseptate, hyaline, rounded at both ends, and 12.5 to 20.0 × 5.0 to 7.5 μm. The morphological characteristics were consistent with descriptions of Colletotrichum species of the C. gloeosporioides species complex, including C. fructicola (Weir et al. 2012). The identity of two representative isolates (PinkL2 & PinkL3) from different apples was confirmed by means of multi-locus gene sequencing. Genomic DNA was extracted using the LGC Mag Plant Kit (Berlin, Germany) in combination with the Kingfisher method (Waltham, USA). Molecular identification was conducted by sequencing the ITS1/ITS4 region and partial sequences of four other gene regions: chitin synthase (CHS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), and beta-tubulin (TUB). The sequences have been deposited in GenBank under accession numbers MT421924 & MT424894 (ITS), MT424612 & MT424613 (CHS), MT424616 & MT424617 (GAPDH), MT424614 & MT424615 (ACT), and MT424620 & MT424621 (TUB). MegaBLAST analysis revealed that our ITS sequences matched with 100% identity to Colletotrichum fructicola (Genbank JX010177). The CHS, GAPDH, ACT and TUB sequences of both isolates were 100% identical with C. fructicola culture collection sequences in Genbank (JX009807, JX009923, JX009436 and JX010400, respectively), confirming the identity of these isolates as C. fructicola. Koch's postulates were performed with 10 mature ‘Pink Lady’ apples. Surface sterilized fruit were inoculated with 20 μl of a suspension of 105 conidia ml–1 after wounding with a needle. The fruits were incubated at 20˚C at high relative humidity. Typical symptoms appeared within 4 days on all fruit. Mock-inoculated controls with sterile water remained symptomless. The fungus was reisolated and confirmed as C. fructicola by morphology and sequencing of all previously used genes. Until recently the reported causal agents of bitter rot of apple in Europe belong to the Colletotrichum acutatum species complex (Grammen et al. 2019). C. fructicola, belonging to C. gloeosporioides species complex, is known to cause bitter rot of apple in the USA, Korea, Brazil, and Uruguay (Kim et al. 2018; Velho et al. 2015). There is only one report of bitter rot associated with C. fructicola on apple in Europe (France) (Nodet et al. 2019). However, C. fructicola is also the potential agent of Glomerella leaf spot (GLS) of apple (Velho et al. 2015; 2019). To the best of our knowledge this is the first report of C. fructicola on apples in Italy. It is important to stress that the C. gloeosporioides species complex is still being resolved and new species on apple continue to be identified, e.g. C. chrysophilum that is very closely related to C. fructicola (Khodadadi et al. 2020). Given the risks of this pathogen the presence of C. fructicola in European apple orchards should be assessed and management strategies developed.

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First Report of Colletotrichum acutatum Causing Bitter Rot on Apple in Italy

Plant Disease ◽

10.1094/pdis-06-11-0483 ◽

2012 ◽

Vol 96 (1) ◽

pp. 144-144 ◽

Cited By ~ 12

Author(s):

M. Mari ◽

M. Guidarelli ◽

C. Martini ◽

A. Spadoni

Keyword(s):

Morphological Characteristics ◽

Weather Conditions ◽

Universal Primers ◽

Colletotrichum Acutatum ◽

Pcr Analysis ◽

The European Union ◽

Single Spore ◽

First Report ◽

Bitter Rot ◽

Wide Range

Italy could be considered the main apple-producing country in the European Union. Italian apple (Malus domestica L. Borkh.) production is estimated at approximately 2.1 million tons and encompasses a wide range of cultivars, harvested from August to November. Colletotrichum acutatum, which causes severe losses to strawberry production, was a regulated organism for all European countries until 2008, when it was removed from the EPPO quarantine pathogen list because of its wide distribution in strawberry-production areas. During the growing season of 2010, fungi were isolated from apple fruits exhibiting bitter rot symptoms after 4 months of storage in several packinghouses in the Emilia Romagna Region. The apples belonged to the Golden Delicious, Granny Smith, Pink Lady and Crisp Pink cultivars. Lesions on the fruit surface were circular and 1 to 3 cm in diameter. When lesions enlarged, they became sunken with relatively firm rotten tissues. The fungal fruiting structures, acervuli, were distributed sparsely or densely on old lesions, and under humid conditions, they discharged an orange conidial mass. Conidia observed with a light microscope appeared hyaline and fusiform, 8 to 16 × 2.5 to 4 μm, with two pointed ends or one rounded end. The fungal isolates were grown on potato dextrose agar (PDA) plates incubated at 25°C. After 7 days, colonies were white, becoming gray to pale orange, and when viewed from the reverse side, the color ranged from pink to reddish orange. Both cultural and morphological characteristics of the pathogen were similar to those described for C. acutatum J.H. Simmonds (3), which is responsible for bitter rot of apple (2). Koch's postulates were performed with one representative isolate from each host by artificial inoculation of 30 healthy apples from the cultivars listed above. Fruit surfaces were disinfected with 70% ethanol, wounded with a sterile needle, and then inoculated with 20 μl of a spore suspension (105 conidia ml–1) prepared from a 15-day-old culture on PDA. Inoculated fruits were sealed in a plastic bag and incubated at 25°C for 10 days. In 92% of fruits, symptoms appeared 10 days later, forming lesions with cream-to-salmon pink fruiting structures. The fungus was reisolated onto PDA from the lesions on the inoculated apples. After 7 days of incubation, the colonies and the morphology of conidia were the same as those of the original isolates. The tests were performed on all four cultivars with similar results. The PCR analysis, carried out using universal primers ITS1 and ITS4 (4) directly from single-spore-derived mycelium (1), resulted in an amplification product with 100% sequence homology with C. acutatum isolate AB626881 from GenBank database. Considering the results obtained, to our knowledge, this is the first report of C. acutatum in Italy causing bitter rot on apple. The disease is common in practically all countries where apples are commercially grown and since the losses could be severe under prolonged warm and wet weather conditions, C. acutatum could represent a serious issue for the Italian apple industry. References: (1) M. Iotti and A. Zambonelli. Mycol. Res. 110:60, 2006. (2) A. L. Jones et al. Plant Dis. 80:1294, 1996. (3) B. C. Sutton. Page 1 in: Colletotrichum: Biology, Pathology and Control. Brit. Soc. Plant Pathol. Oxon. UK 1992. (4) T. J. White et al. Page 315 in: PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

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SARS-CoV-2 within-host diversity and transmission

Science ◽

10.1126/science.abg0821 ◽

2021 ◽

Vol 372 (6539) ◽

pp. eabg0821 ◽

Cited By ~ 1

Author(s):

Katrina A. Lythgoe ◽

Matthew Hall ◽

Luca Ferretti ◽

Mariateresa de Cesare ◽

George MacIntyre-Cockett ◽

...

Keyword(s):

United Kingdom ◽

Severe Acute Respiratory Syndrome ◽

Phylogenetic Tree ◽

Immune Escape ◽

Clinical Samples ◽

Diversity Patterns ◽

Host Diversity ◽

Viral Loads ◽

The United Kingdom ◽

Low Levels

Extensive global sampling and sequencing of the pandemic virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have enabled researchers to monitor its spread and to identify concerning new variants. Two important determinants of variant spread are how frequently they arise within individuals and how likely they are to be transmitted. To characterize within-host diversity and transmission, we deep-sequenced 1313 clinical samples from the United Kingdom. SARS-CoV-2 infections are characterized by low levels of within-host diversity when viral loads are high and by a narrow bottleneck at transmission. Most variants are either lost or occasionally fixed at the point of transmission, with minimal persistence of shared diversity, patterns that are readily observable on the phylogenetic tree. Our results suggest that transmission-enhancing and/or immune-escape SARS-CoV-2 variants are likely to arise infrequently but could spread rapidly if successfully transmitted.

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Clustering of equine grass sickness cases in the United Kingdom: a study considering the effect of position-dependent reporting on the space–time K-function

Epidemiology and Infection ◽

10.1017/s0950268804003322 ◽

2004 ◽

Vol 133 (2) ◽

pp. 343-348 ◽

Cited By ~ 31

Author(s):

N. P. FRENCH ◽

H. E. McCARTHY ◽

P. J. DIGGLE ◽

C. J. PROUDMAN

Keyword(s):

United Kingdom ◽

Management Practices ◽

Space Time ◽

Pasture Management ◽

Local Climate ◽

Temporal Properties ◽

Time Interaction ◽

The United Kingdom ◽

Grass Sickness ◽

Type C

Equine grass sickness (EGS) is a largely fatal, pasture-associated dysautonomia. Although the aetiology of this disease is unknown, there is increasing evidence that Clostridium botulinum type C plays an important role in this condition. The disease is widespread in the United Kingdom, with the highest incidence believed to occur in Scotland. EGS also shows strong seasonal variation (most cases are reported between April and July). Data from histologically confirmed cases of EGS from England and Wales in 1999 and 2000 were collected from UK veterinary diagnostic centres. The data did not represent a complete census of cases, and the proportion of all cases reported to the centres would have varied in space and, independently, in time. We consider the variable reporting of this condition and the appropriateness of the space–time K-function when exploring the spatial-temporal properties of a ‘thinned’ point process. We conclude that such position-dependent under-reporting of EGS does not invalidate the Monte Carlo test for space–time interaction, and find strong evidence for space–time clustering of EGS cases (P<0·001). This may be attributed to contagious or other spatially and temporally localized processes such as local climate and/or pasture management practices.

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First Report of Pythium aphanidermatum Causing Stalk Rot on Abelmoschus manihot (L. ) Medic in China

Plant Disease ◽

10.1094/pdis-06-21-1133-pdn ◽

2021 ◽

Author(s):

Qing Qu ◽

Liu Shiwei ◽

Ning Liu ◽

Yunxia Liu ◽

Jia Hui ◽

...

Keyword(s):

Management Practices ◽

Vascular System ◽

Morphological Characteristics ◽

Pythium Aphanidermatum ◽

Negative Control ◽

Perennial Herb ◽

Universal Primers ◽

First Report ◽

Stalk Rot ◽

Aerial Hyphae

Abelmoschus manihot (Linn. ) Medicus (A. manihot) is an annual to perennial herb of the Malvaceae okra, mainly distributed in Guangdong, Guangxi, Fujian, Hunan, Hubei provinces. It can not only be used as an ornamental flower, but also has important economic and medicinal value. Last year, 10% A. manihot in 1,000 acres were observed with stalk rot in the Zhongshang Agricultural Industrial Park, 50 meters east of Provincial Highway 235 in Gaoyang County of Hebei province. Internal discoloration of the stem began brown to black discoloration of the vascular system and became hollow, with the mycelium growing on the surface. Stems from symptomatic plants (approximately 5 mm2) were dissected, washed free of soil, then soaked in 75% ethanol for 16 s to surface-sterilize, and 40 s in HgCl2, then rinsed three times in sterile water. After being dried with blotting paper, five pieces were placed on potato dextrose agar (PDA). After cultured 2 or 3 days, five isolates were purified and re-cultured on PDA in the dark at 25°C. The color of the colony was white. The hyphae were radial in PDA, and the aerial hyphae were flocculent, well-developed with luxuriant branches. The colonies were white and floccus, and the aerial hyphae were well developed, branched and without septum on corn meal agar (CMA). The sporangia were large or petal shaped, composed of irregular hyphae, terminal or intermediate , with the size of (31.6-88.4) μm ×(12.7- 14.6) μm. Vesicles were spherical, terminal or intermediate, ranging from 14.6 to 18.5μm. Oogonia were globose, terminal and smooth which stipe was straight. Antheridia were clavate or baggy and mostly intercalary, sometimes terminal. Oospores were aplerotic, 21.5 to 30.0 μm in diameter, 1.6 to 3.1 μm in wall thickness. The isolates morphological characteristics were consistent with P. aphanidermatum (van der Plaats-Niterink 1981, Wu et al. 2021 ). To identify the isolates, universal primers ITS1/ITS4 (White et al. 1993) were used for polymerase chain reaction–based molecular identification. The amplification region was sequenced by Sangon Biotech (Shanghai, China) and submitted to GenBank (MW819983). BLAST analysis showed that the sequence was 100% identical to Pythium aphanidermatum. Pathogenicity tests were conducted 3 times, with 4 treatments and 2 controls each time. The plants treated were 6 months old. Then the hyphae growing on PDA for 7 days were cut into four pieces. Next, they were inoculated into the soil of the A. manihot. Negative control was inoculated only with PDA for 7 days ( Zhang et al. 2000). The plants were then placed in a greenhouse under 28°C, 90% relative humidity. After inoculated 20 to 30 days, the infected plants showed stalk rot, the same symptoms as observed on the original plants. The control plants didn’t display symptoms. Pythium aphanidermatum was re-isolated from infected stems and showed the same characteristics as described above and was identical in appearance to the isolates used to inoculate the plants. To our knowledge, this is the first report of Pythium aphanidermatum infecting A. manihot stem and causing stalk rot in China. It may become a significant problem for A. manihot. Preliminary management practices are needed for reducing the cost and losses of production.

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