First report of Southern blight, caused by Athelia rolfsii (syn. Sclerotium rolfsii Sacc.), on Hellebores in North America

Lenten rose (Hellebores hybridus) is an herbaceous perennial grown in landscapes and valued for early spring flowers and high levels of deer resistance. An additional benefit as a landscape plant comes from the high level of disease resistance, with only three fungal pathogens reported in North America. In August of 2021, a Lenten rose plant within a mature landscape in Silver Spring, MD, USA, (lat 39.116629 long 77.043198) was found with a collapsed canopy and brown stems near the soil line. Small clusters of brown sclerotia-like objects were seen along the stem. Samples of the sclerotia and diseased tissue were dipped in 70 percent ethanol for 15 sec, transferred to 5 percent NaClO for 30 sec, immersed in sterile water for one minute, then plated onto Potato Dextrose Agar. Sclerotia-like objects germinated and white mycelia covered the plates within five days of germination. Hyphae emerged from diseased tissue within two days and also grew rapidly. Cultures from sclerotia-like objects and diseased tissue produced white sclerotia which melanized to brown spherical sclerotia ranging in size from two to four mm. Culture samples (1 cm square) were excised from the culture plates and transferred to the base of three two-year old potted hellebore plants. Control plants had blocks of PDA placed at the base of the plants. Plants were placed in plastic bags for two days to maintain humidity, then maintained at room temperature without plastic bags. Petioles turned brown and collapsed within seven days of inoculation. White, fan-like hyphae were present along with maturing sclerotia. Samples from surface sterilized tissue and sclerotia produced the same culture morphology as the originally isolated cultures. Non-inoculated plants remained healthy, and the pathogen was not isolated from non-inoculated plants. Individual DNA samples were prepared from original cultures and the re-isolated cultures. Molecular identification was performed by amplification of the internal rRNA transcribed spacer region (ITS1/4, White et al. 1990 ), the large subunit rRNA (LSU), and the elongation factor-1A (EF1a). Amplification products were cloned into TOPO-TA pcr4 vector and sequenced (Macrogen USA). Sequences were submitted to GenBank for IT1/4 (OK172559) and LSU (OK172560). Homology to ITS1/4 was found with Athelia rolfsii (MN622806), to LSU with Athelia rolfsii (MT225781) and for EF1a with Athelia rolfsii (MW322687). This is the first report of Athelia rolfsii on Hellebores in North America (Farr, D.F & Rossman, A.Y. Fungal Databases, U.S. National Fungus Collections, ARS, USDA. Retrieved September 10, 2021). This report is unique in that few pathogens are known to infect Hellebores(Taylor et al. 2011) and southern blight is not commonly isolated in landscape plantings at Maryland latitudes. 1. White et al. PCR Protocols: A Guide to Methods and Amplifications. Academic Press, San Diego, 1990 2. Taylor, R.K., Romberg, M.K. & Alexander, B.J.R. A bacterial disease of hellebore caused by Pseudomonas viridiflava in New Zealand. Australasian Plant Dis. Notes 6, 28–29, 2011.

Presence of Powdery Mildew Caused by Erysiphe corylacearum on Hazelnut (Corylus avellana) in Italy

Hazelnut (Corylus avellana) is widely grown in Italy, which is the second largest producer worldwide with 132,700 tonnes harvested from 78,593 hectares (FAOSTAT, 2018 ). Powdery mildew caused by Phyllactinia guttata has been reported in Italy and in other European countries, but recently in Austria, Switzerland and in central Europe a new species was discovered (Voglmayr et al., 2020; Beenken, 2020). During summer 2020, in Villar Fioccardo (Torino province, Piedmont, Italy) on hazelnut (cv. ‘Tonda Gentile’) growing on the edges of private gardens and parks, an extensive colonization of the adaxial side of the leaves with white powdery mycelium covering more than 80% of the surface was observed. Also, the abaxial side of the leaves showed the scattered presence of powdery, white, and thin mycelium. The powdery fungal pathogen collected from leaves had amphigenous, hyaline, branched, septate 1.5 to 3.7 μm wide mycelium; lobed, solitary hyphal appressoria; vertically elevated above the mycelium 53 to 82 μm long and 5 to 12 μm wide conidiophores (n = 30); hyaline, ellipsoid, ovoid to doliform conidia, solitary on conidiophores, 21 to 36 μm long, 15 to 21 μm wide (average 28 to 18 μm) (n = 50). Chasmothecia appeared in late September 2020 and they were spherical, single or in groups, 83 to 138 (average 100) μm in diameter (n = 50); 7 to 15 aseptate appendages were straight, sometimes flexuous, 55 to 111 (average 73) μm long (n = 50), with four to five times dichotomous branched apexes and recurved tips. In each chasmothecium, there were three to five ellipsoid, ovoid to subglobose asci with a length of 41 to 60 μm and a width of 28 to 56 μm (average 52 to 44 μm) (n = 30). Asci contained four to eight ascospores, 15 to 26 μm long and 10 to 17 μm wide (average 19 to 12 μm) (n =50). Mycelia were carefully scraped from the leaves with a scalpel and DNA was extracted by using the E.Z.N.A. Fungal DNA Mini Kit (Omega Bio-Tek, Darmstadt, Germany). Partial rDNA internal transcribed spacer region (ITS) of two isolates (DB20SET01, DB20SET01) was amplified using specific primers PMITS1/PMITS2 (Cunnington et al. 2003) and sequenced. Obtained sequences were deposited in GenBank (Accession Nos. MW045425, MW045426). BLAST analysis of the obtained 749-bp fragments showed 100% identity to ITS rDNA sequences of Erysiphe corylacearum from Switzerland (MN822721) and Azerbaijan (LC270863). One-year-old plants of C. avellane cv. Tonda Gentile were artificially inocuated by dusting conidia from infected leaves. Inoculated plants were incubated under controlled conditions at 23°C ± 1 and 70 to 80% relative humidity. Typical symptoms (white bloom) appeared on the upper surface of the leaves at 8 to 10 days after inoculation. No symptoms were found on control plants treated with sterile water. The fungus isolated from inoculated leaves was morphologically identical to the original isolates from diseased plants collected from Villar Fioccardo. Erysiphe corylacearum causes a new and aggressive form of powdery mildew. Since the first observation in north-eastern Turkey in 2013, it has spread rapidly throughout the Black Sea region, causing significant economic losses (Sezer et al., 2017). It has also been reported in Iran, Azerbaijan, and Ukraine (Arzanlou et al. 2018; Heluta et al., 2018). The disease has been observed sporadically in Piedmont, Italy, during summer 2020 (Regione Piemonte & Agrion, 2020) in some hazelnut growing areas, but presently, doesn’t appear to impact yield. This is the first report of E. corylacearum, causing an aggressive powdery mildew on hazelnut in Italy, and as such, may more severely affect hazelnut groves in Italy and cause considerable yield losses. Literature cited Arzanlou M et al. 2018. Forest Pathology, 48:e12450. https://doi.org/10.1111/efp.12450. Beenken L et al. 2020. New Disease Reports 41, 11. http://dx.doi.org/10.5197/j.2044-0588.2020.041.011. Cunnington JH et al. 2003. Australasian Plant Pathology, 32, 421-428. Food and Agriculture Organization (FAO). 2018. http://www.fao.org/faostat/en/#home Heluta V.P. et al.2019. Ukrainian Botanical Journal, 2019, 76(3), 252-259. Regione Piemonte SFR & Agrion. 2020. https://www.regione.piemonte.it/web/sites/default/files/media/documenti/2020-10/mal_bianco_nocciolo_da_erysiphe_corylacearum.pdf Sezer AD et al. 2017. Phytoparasitica, 45, 577-581. Voglmayr H et al. 2020. New Disease Reports, 42, 14 http://dx.doi.org/10.5197/j.2044-0588.2020.042.014

Discovery of Cyclotides from Australasian Plants

This article is part of a special issue celebrating the contributions of Professor Paul Alewood to peptide science. We begin by providing a summary of collaborative projects between the Alewood and Craik groups at The University of Queensland and highlighting the impacts of some of these studies. In particular, studies on the discovery, synthesis, structures, and bioactivities of disulfide-rich toxins from animal venoms have led to a greater understanding of the biology of ion channels and to applications of these bioactive peptides in drug design. The second part of the article focuses on plant-derived disulfide-rich cyclic peptides, known as cyclotides, and includes an analysis of the geographical distribution of Australasian plant species that contain cyclotides as well as an analysis of the diversity of cyclotide sequences found in Australasian plants. This should provide a useful resource for researchers to access native cyclotides and explore their chemistry and biology.

Virtual Issue in Australian Journal of Botany: rare and threatened plant conversation and recovery

Virtual Issues consist of previously published papers that are repackaged into an online themed collection. With appropriate marketing, and making them free for a limited time, these issues are a powerful tool that allow all readers to rediscover and access content already published. Release of this Virtual Issue of Australian Journal of Botany was timed to coincide with the 11th Australasian Plant Conservation Conference (APCC11), held on 14–18 November at the Royal Botanic Gardens, Melbourne. For more information, please visit: http://www.publish.csiro.au/bt/content/VirtualIssues.

Recent Emergence of the Mild Strain of Tomato yellow leaf curl virus as a Cause of Tomato Yellow Leaf Curl Disease of Processing Tomatoes (Solanum lycopersicon) in the Dominican Republic

In the early 1990s, the monopartite begomovirus Tomato yellow leaf curl virus (TYLCV) was introduced into the Dominican Republic (DO), and molecular characterization revealed it was an isolate of TYLCV-Israel (TYLCV-IL[DO]) (3,5). In 2006, a study of the variability of TYLCV in DO revealed that TYLCV-IL[DO] was associated with all samples of tomato yellow leaf curl (TYLC) tested and, thus, that the virus had been genetically stable for >15 years (2). However, in 2010 and 2011, 2 of 10 and 11 of 18 samples of TYLC, respectively, were negative for TYLCV infection based upon PCR with the TYLCV-specific primer pair, 2560v (5′-GAGAACAATTGGGATATG-3′)/1480c (5′-AATCATGGATTCACGCAC-3′), which directs the amplification of a ~1.7 kb fragment. In 2011, two such samples from the Azua Valley were tested by PCR with the 1470v (5′-AGTGATGAGTTCCCCTGTGC-3′)/UPC2 primer pair (1), and sequence analysis of the ~0.4 kb fragment amplified from both samples revealed infection with the mild strain of TYLCV (TYLCV-Mld). A primer specific for TYLCV-Mld was designed (2070v, 5′-AAACGGAGAAATATATAAGGAGCC-3′), and PCR with the 2070v/1480c primer pair directed the amplification of the expected ~2.1 kb fragment from all 11 TYLC samples collected in 2011 that were PCR-negative for TYLCV-IL[DO] infection. Sequence analyses confirmed these were TYLCV-Mld fragments. The complete TYLCV-Mld genome was amplified from two samples from the Azua Valley with Templiphi, the amplified DNA products digested with Sal I, and the resulting ~2.8 kb fragments ligated into Sal I-digested pGEM-11. The complete sequences of these isolates were 2,791 nt and 99% identical to each other and 98% identical to sequences of TYLCV-Mld isolates. The TYLCV-Mld isolates from the DO were designated TYLCV-Mld:DO:TY5:01:2011 (KJ913682) and TYLCV-Mld:DO:TY5:02:2011 (KJ913683). A multimeric clone of TYLCV-Mld:DO:TY5:01:2011 was generated in the binary vector pCAMBIA1300 by cloning a 2.2 kb Sal I-EcoRI fragment containing the intergenic region to generate a 0.8-mer (pCTYMld0.8), and then the full-length Sal I fragment was cloned into the Sal I site of pCTYMld0.8 to generate a 1.8-mer (pCTYMldDO-01-1.8). Tomato plants agroinoculated with Agrobacterium tumefaciens carrying pCTYMldDO-01-1.8 developed severe TYLC disease symptoms 10 to 14 days after inoculation, whereas plants inoculated with a strain carrying the empty vector did not develop symptoms. Samples of processing tomatoes with TYLC were collected in 2012 to 2014 in the DO and tested for TYLCV-IL[DO] and TYLCV-Mld by PCR with the 2560v/1480c and 2070v/1480c primers pairs, respectively; these samples had infections of 93% (13/14), 86% (18/21), and 61% (11/18) with TYLCV-Mld; 29% (4/14), 19% (4/21), and 56% (10/18) with TYLCV-IL[DO]; and 21% (3/14), 5% (1/21), and 28% (5/18) with both viruses, respectively. These results reveal that there has been a striking population shift in the begomovirus causing TYLC in the DO, with TYLCV-Mld becoming predominant. This may reflect selection pressure(s) favoring a small pre-existing population of TYLCV-Mld, such as new tomato varieties, or a recent introduction event, such as that described in Venezuela (4). References: (1) R. W. Briddon and P. G. Markham. Mol. Biotechnol. 1:202, 1994. (2) R. L. Gilbertson et al. Page 279 in: Tomato yellow leaf curl virus disease. Springer, 2007. (3) M. K. Nahkla et al. Plant Dis. 78:926, 1994. (4) G. Romay et al. Australasian Plant Dis. Notes, in press, 2014. (5) R. Salati et al. Phytopathology 92:487, 2002.