Scientific Papers. Series B, Horticulture. Vol. LXIII, No. 1, 2019 Print ISSN 2285-5653, CD-ROM ISSN 2285-5661, Online ISSN 2286-1580, ISSN-L 2285-5653

LENTICEL ROT, SPP. IN ROMANIA. NEW REPORTS INFOGRAPHIC

Roxana CICEOI1, Beatrice IACOMI2

1Research Center for Studies of Food and Agricultural Products Quality, University of Agronomic Sciences and Veterinary Medicine, Bd. Marasti, No 59, Sector 1, Bucuresti, România 2Plant Sciences Department, University of Agronomic Sciences and Veterinary Medicine, Bd. Marasti, No 59, Sector 1, Bucuresti, România ,

Corresponding author email: [email protected]

Abstract

Neofabraea spp., also known as bull`s eye rot, lenticel rot, gloeosporium rot or bitter rot, are one of the main true post- harvest fungal pathogens affecting apples and pears storage in Europe, North America, Australia and some countries from South America. In Europe, considerable losses occur in the Western part of the continent, for some susceptible varieties, mainly in the regions with high rain fall in the last part of the growing season. Although the symptoms become visible after a few months of storage, the fruits are infected in the orchard, as this is a latent infecting agent, causing a true post-harvest disease. Due to different taxonomic opinions over time, Neofabraea spp. were usually considered as Gloeosporium sp., or sometimes as the perfect form of this species. In fact, there are 15 different species of Neofabraea and 47 species of Gloesporium registered in mycobank or Index Fungorum. Pre-harvest fungicide treatments, if properly applied, can partially control the disease, but as synthetic products are likely to leave residues, the number of interventions tends to be reduced and the use of certain substances is often limited. Different non-synthetic pre-harvest treatments have been tested against Neofabraea spp., as bicarbonate, with low results in the susceptible areas and hot water treatments after harvest gave some promising results, but the method has to be tested on each area and each variety for a proper use. In Romania, the presence of the fungus is often hidden by the infections with Penicillium species, which lead to apple decay long before the gloeosporium rot become visible. As post-harvest advanced cold storage facilities became common in our country in the last decade, the latent pathogens can now be identified. Our infographic aims to evidence of the presence of Neofabrae spp. in Romania and open a new path of study in the post-harvest fungal pathogens.

Key words: Neofabraea spp., gloeosporium rot, apple, postharvest disease, cold storage.

INTRODUCTION Monilia, Neonectria, Neofabraea, and Penicillium genus (Köhl et al., 2018; Simtniece Nowadays losses during storage are one of the et al., 2017; Wenneker and Köhl, 2014). While main concerns for fruit producers, as 15% to Monilia spp., Botrytis spp. and Penicillium spp. 60% (Naets et al., 2018) of stored fruits may be originate from infections from wounds during lost before marketing, depending on the harvest and handling operations, usually being weather conditions from the respective year, called ”wound pathogens”, Neofabraea spp., crop technology, variety, but also the storage Colletotrichum spp. and Gloeosporium spp. are conditions. Around 30% losses are most likely fungi infecting plants and fruits in the orchards, due to microbial decay (Naets et al., 2018). during the growing season. The fungus remain Apple is one of the fruits with the longest latent for few months of storage, thus being period of storage required by the market and responsible for ”latent infections” or known as the apple rot is the most important cause of a ”true” storage pathogens (Simtniece et al., losses during storage. The second main cause 2017; Wenneker and Köhl, 2014). of storage losses, the functional or non- Gloeosporium rot, also refered as Bull’s eye parasitic breakdowns (Wilkinson, 1954) has rot, lenticel rot or bitter rot (Köhl et al., 2018; been solved during time be apple breeding and Michalecka et al., 2016), is an important improving the storage conditions. The most postharvest disease of apples and pears, important pathogens that lead to apple decay occurring worldwide in the major fruit growing during storage belong to Alternaria, Botrytis, areas of Pacific Northwest of US, Canada, Colletotrichum, Fusarium, Gloeosporium, Chile, Australia, New Zeeland and Europe 161 (Jayawardena et al., 2019; Köhl et al., 2018; During time, Neofabraea (anamorph Michalecka et al., 2016; Cunnington, 2004). It Phlyctema) was long debated, and still is, but also cause anthracnose canker and perennial according to updated nomenclature (Index canker on pome trees, canker on Populus sp., Fungorum, 2019), the genus includes 14 coin canker of ash, fruit rot on kiwifruit, fruit species, of which four species have been spot on olive and leaf spot on citrus associated with bull`s eye rot on apples: (Jayawardena et al., 2019). Taxonomically, it Neofabraea vagabunda (Desm.) Rossman belongs to , , (synonyms: N. alba Jacks, Pezicula alba; Dermataceae (Jayawardena et al., 2019). Gloeosporium album), N. perennans Kienholz In Europe, it has been reported as the main (syn. P. perennans), N. malicorticis Guthrie cause of decay during storage in Norway, (syn. P. malicorticis) and N. kienholzii Spotts, Germany, France (Saville, 2015), and its Lévesque & Seifert (syn. Cryptosporiopsis presence was confirmed in Netherlands (Köhl kienholzii). Bogo et al, 2018 found that et al., 2018), Poland (Michalecka et al., 2016), Neofabraea actinidiae and N. brasiliensis were UK (Kingsnorth et al., 2017), Italy (Cameldi et the causal agents of apple bull’s-eye rot in al, 2017). The pathogen is a true post-harvest southern Brazil, a report that need further pathogen (Wenneker and Köhl, 2014), that confirmation. A detailed history of taxonomic develops after three-four months on the cold evolution is presented in the update with stored apples or pears (Michalecka et al., molecular phylogeny done by Jayawardena et 2016). In the orchard, the fungus infects the al., 2019 and Marin-Felix et al., 2017. woody tissues of the apple and pear trees N. alba is the most spread gloeosporium rot (Michalecka et al., 2016, Wenneker and Köhl, agent for apples and pears in continental 2014), causing cankers on branches and twigs Europe, but it was reported also in eastern (perennial canker). It may also develop as Canada, eastern North America, in the USA saprophytic fungus, on dead wood or pruning Pacific Northwest, Australia and Chile snags (Michalecka et al., 2016), or stay in the (Michalecka et al., 2016). weeds, acting as a source of inoculum for the N. perennans was usually reported in humid next year infections (). According to areas of the west coast of the USA, in the Kingsnorth et al., 2017, during the last century Washington region, in western Canada and Neofabraea spp. was the main cause of apple Australia (Michalecka et al., 2016), but also in rot in some EU countries, with rise and falls on China (Cao et al., 2010). In Europe has been its yearly prevalence (Saville, 2015). For detected in the Netherlands, Germany and the instance, in UK, a survey regarding the fungal UK (Köhl et al., 2018). rots recorded in cold stored apples performed N. malicorticis was spotted on the west coast of in 1920 mentions Gloeosporium as one of the the USA and Canada, in New Zealand and in less present diseases while the same type of Denmark, the Netherlands, and Portugal survey repeated in 1937-1939 describes the (Michalecka et al., 2016). bitter rots produced by Gloeosporium album N. kienholzii was detected in samples from and G. perennans as the most important decay Nova Scotia, Canada, and Portugal causes, at a level of 15.41% and 8.16% for (Michalecka et al., 2016). 1937 and 1938 year respectively (Wilkinson, According to Jayawardena et al., 2019, 1954). In 1960, losses due Neofabraea spp. identification of Neofabraea spp. based solely went up to 50% for Cox variety while more on morphological characters is not encouraged, recently, losses up to 35% have been reported due to high similarity with other species. The (Saville, 2015). The post-harvest control most reliable identification methods are based measures with fungicides and advances in on molecular means (Lin et al, 2018; Cao et al, storage technologies made losses negligible at 2010, de Jong et al., 2001), TUB2 gene being the end of last century, but recently susceptible the best single genetic marker for the genus varieties started to be affected again by this Neofabraea, and combined with ITS, LSU, fungus and the confirmation of Neofabraea RPB2 and TUB2 genetic markers, all species spp. was done by molecular means (Saville, can be differentiate. 2015; Kingsnorth et al., 2017).

162 In Romania, the presence of the fungus is often MATERIALS AND METHODS hidden by the infections with Penicillium spp., which lead to apple decay long before the Stored apples from three different counties gloeosporium rot become visible. One from Romania (Arad, Ilfov and Bucharest) reference to Neofabraea in Romania is made were sampled to detect the presence of by Florian et al., 2018, but only as the Neofabraea spp., five months after storage. teleomorph state of Gloeosporium, considered Additionally, two apples from the market were the anamorph state. According to Index randomly selected, to test if Neofabraea spp. Fungorum, (2019), the taxonomy links between symptoms were easily recognised. In total, 60 the two genera is complex and morphological organic apples and 41 conventional apples, and molecular identification is prior needed. belonging to 10 different varieties - Rubinola, Neofabraea spp. are predominantly controlled Topaz, Renoir, Gemini, Red Prince, Golden with copper-based fungicides (Garton et al., Delicious, Starkrimson, Generos, Idared, Gala, 2019) Pre-harvest treatments using chemical were assessed. fungicide can control Neofabraea spp. in some All apples from Bucharest and Arad were extent, but due to high level of residues that can grown in organic system. The apples were remain on the fruits and other associated risks stored either in cold storage or controlled (Aguilar, 2018), if the treatment is done just atmosphere (5 or 10% CO2) and removed from before the harvest, the number of interventions the storage when decay symptoms started to must decrease and the possibility to use some become visible. active substances is limited. Organic or non- Prior to PDA culture, surface disinfection was synthetic pre-harvest treatments have been performed. To isolate the fungal agent, fruit tested against Neofabraea spp., as bicarbonate, lesions were sprayed with 70% ethanol, let to but in the susceptible areas the results are dry, than the peel was removed aseptically with unsatisfactory. Daniel et al., 2015 mention the a scalpel and the rotted and healthy tissue under use of garlic extract and clove oil for the lesion (at the limit of rotted area – fig. 1) postharvest treatments against Neofabraea spp. was isolated and placed onto Potato Dextrose Treatments with hot water immediately after Agar (PDA), the plates being incubated at 20°C harvest gave some promising results, but the in the dark (Wenneker, et al., 2017), in IN 110 method has to be tested on each area and each (Memmert) incubators. The disinfection with variety for a proper use (Di Francesco et al., ethanol aimed mainly to prevent contamination 2018; Neri et al., 2009). Therefore, early with Penicillium spp. or other saprophytic identification of the inoculum in the orchard species. The rot on apples usually appears as and applying preventive methods is the most circular lesion, flat to slightly sunken, light suitable solution. Aguilar et al., 2018 highlights brown to dark brown with a light coloured the importance of proper pruning, canker centre on fruits (Jayawardena et al., 2019). removal and aphid control in orchards. The colonies were analysed at 30 days after As post-harvest advanced cold storage facilities isolation on PDA. became common in Romania in the last decades, the latent pathogens can now be identified. The most affordable way to confirm the presence of a Neofabraea spp. following symptoms identification on apples is the PDA culture. As only preliminary data were obtained, it was preferred the form of an infographic as first report (Abilock and Williams, 2014). The present work aims to trigger the attention of all interested stakeholders about the presence of Neofabraea spp. in Romania and open a new path of study in the post-harvest fungal pathogens. Figure 1. Prelevation of rotten pulp after fruit surface disinfection 163 RESULTS AND DISCUSSIONS

Out of the 99 analysed fruits from Romania, 14 isolates of Neofabraea spp. were obtained. The colony morphology was compared with available data from literature (Kingsnorth et al., 2017; Chen et al., 2016; Saville, 2015; Spotts et al., 2009) and with the pure cultures of isolates from Poland, InHort, obtained through the Euphresco project Earldetec. Out of the 59 apples harvested from the experimental field of University of Agronomic Sciences and Veterinary Medicine (USAMV) Bucharest, four apples lead to five Neofabraea isolates, belonging to Rubinola (2), Gemini (1) and Topaz (1) varieties (fig. 2).

Figure 3. Neofabraea sp. isolates of apples from Moara Domneasca cold storage, Ilfov County (apples, Petri dish face view, Petri dish back view)

Figure 2. Neofabraea sp. isolates from experimental orchard of USAMV Bucharest (apples, Petri dish face view, Petri dish back view)

From Ilfov county, Moara Domneasca experimental farm, out of the 39 apple samples, three lead to the selection of four isolates of Figure 4. Neofabraea sp. isolates of apples Neofabraea spp. The host varieties were Idared from Horia Village, Arad County (apples, Petri dish face and Ionathan (fig. 3). view, Petri dish back view) 164 From Arad County, Horia village commercial project “Early detection of apples fungal farm, out of the 11 samples, three Golden pathogens”. Delicious apples lead to the selection of five isolates of Neofabraea spp. (fig. 4). REFERENCES

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