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Article

Brenneria nigrifluens Isolated from Aesculus hippocastanum L. Bark in Hungary

by
Imola Tenorio-Baigorria
1,2,
Gergely Botyánszki
1,
Rita Gyuris
1,
György Zsigó
3,
László Palkovics
4,* and
Anita Végh
1
1
Institute of Plant Protection, Buda Campus, Hungarian University of Agriculture and Life Sciences, 1118 Budapest, Hungary
2
National Food Chain Safety Office, Directorate of Food Chain Safety Laboratory, Plant Health Diagnostic National Reference Laboratory, 1118 Budapest, Hungary
3
Hungarian Chamber of Crop Protection Specialists and Plant Doctors, 1037 Budapest, Hungary
4
Department of Plant Sciences, Faculty of Agriculture and Food Sciences, Széchenyi István University, 9200 Mosonmagyaróvár, Hungary
*
Author to whom correspondence should be addressed.
Forests 2022, 13(2), 227; https://doi.org/10.3390/f13020227
Submission received: 27 December 2021 / Revised: 26 January 2022 / Accepted: 29 January 2022 / Published: 2 February 2022
(This article belongs to the Section Forest Health)
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Abstract

:
In Hungary, from the beginning of the 19th century, horse-chestnut trees have been planted widely and are popular ornamental trees in public parks, along streets, and in gardens. In the summer of 2015, longitudinal cracks on the trunk and branches and the intensive oozing of brown liquid were observed from a wound in a horse-chestnut tree in a park in Budapest. Some years later, in 2018 and 2019, the same symptoms were found in trees in other locations in Budapest. Several bacteria were reported that induce similar symptoms, including cracks and cankers on the bark of trunks and branches and sticky, white, red, brown, or black oozing. These pathogens belong to the genera Brenneria and Lonsdalea. Bark and exudate samples were taken with the aim of identifying the causal agent by conventional and molecular methods. Our results confirmed that the bacteria isolated from Aesculus hippocastanum trees belong to the genus Brenneria and phylogenetic analysis of the 16S rRNA gene region proved to have the closest phylogenetic relation with the Brenneria nigrifluens strains.

1. Introduction

The horse chestnut was previously mentioned as one of the most significant urban trees in the cities of Hungary [1]. The Hungarian Ornamental Horticulturist Association publishes a list of urban trees every two years, which reports much valuable information (e.g., tolerance to urban environments, major pests). Aesculus hippocastanum L. has not been included since 2018, since it is sensitive to the dry and warm climate of the cities, to air pollution, and, particularly, to de-icing salting [2].
Within a Hungarian game reserve there is a horse-chestnut stand (22.5 hectares), located in Gyarmatpuszta, that was established at the end of the 19th century [3]. The purpose was to provide food for wild animals during autumn and winter without incurring any damage to oak regrowth caused by them—big game often causes severe damage when looking for food. In these forests, such game can no longer find the feed it previously had in its natural forest stands (primarily acorn and beechnut), with which they stabilized themselves before the harsh winter [3,4]. Among others, the fruits of horse chestnuts and acorns are significant for them [5]. Therefore, horse-chestnut stands, which usually produce their crops every year, are best suited for feeding big game. Besides, the horse chestnut has a higher nutritional value than acorn, and is also preferable to these animals—especially to deer [3,4]. However, experts very often have a negative attitude toward forests that have been established from non-native tree species; yet Lakatos et al., in their study, pointed out that forests planted with horse chestnuts do not have significantly lower diversity of birdlife and soil fauna than in its natural state. Eighteen, 16, and 14 bird species were found associated with isolated chestnut stands and hornbeam and sessile oak stands, respectively. In the soil of the studied area, 45 species of Collembola were found, a high population of species. Based on the Collembola species investigated, no significant differences were found between hornbeam–sessile oaks and alien horse-chestnut stands in terms of number of species or individuals inhabiting them. The first occurrence of Jevania weinerae, in 14 horse-chestnut soil samples in Hungary, was a faunistic specialty; this species was previously known only to riparian alder forests in Poland [6].

1.1. Members of Brenneria Genus

The genus Brenneria belongs to the family Pectobacteriaceae (Gammaproteobacteria, Enterobacterales), which contains four further genera: Pectobacterium, Dickeya, Lonsdalea and Sodalis [7]. Members of the Brenneria genus affect several deciduous tree species (Table 1) and cause similar symptoms, including cracks; shallow or deep cankers on the trunk and branches; white, black, or brown, watery, or sticky substance oozing on the bark; and, in some cases, wilting and drying [8,9,10,11,12].
The Brenneria genus contains eight species and four subspecies. However, these species once belonged to the genus Erwinia. The first species was described as Erwinia salicis by Day (1924) as causing watermark disease on willow [13]. In 1957, Wilson et al. reported a new bacterial disease, shallow bark cankers on Persian walnut trees, that was caused by Erwinia nigrifluens [8]. Ten years later, deep bark cankers in walnut (Erwinia rubrifaciens) and drippy nut disease in oak (Erwinia quercina) were described [27,29]. Erwinia alni was published as the causal agent of bark canker on alder trees [9]. In 1998, based on molecular methods, six Erwinia species were transferred to the Brenneria genus: B. alni, B. nigrifluens, B. quercina, B. paradisiaca, B. rubrifaciens, and B. salicis [36]. Later, in 2012, Brady et al. reclassified Brenneria quercina and proposed a new genus, Lonsdalea [37]. Two further Brenneria species were described that are associated with acute oak decline (AOD), B. goodwinii and B. roseae [31,33], and two species were isolated from Populus × euramericana bark cankers (B. populi, B. corticis) [12,35]. From the above-mentioned species, B. salicis and B. nigrifluens are reported in Hungary as well [16,22]. Watermark disease in willow was first described in the counties Győr, Moson, and Sopron and significant tree death was observed [16]. In 2013, cracks and black ooze appeared on the bark of walnut trees, from which Brenneria nigrifluens was isolated and found to be the causal agent [22].

1.2. Diseases Associated with Bleeding Canker on the Bark of Horse-Chestnut Trees

Two pathogens are known to cause bleeding canker disease on Aesculus hippocastanum. Since the 1970s, bark necrosis and gummy substance exudation have been observed from the bark of horse-chestnut trees. Further symptoms are crown dieback, water-soaked lesions under the necrotised bark on the trunk, and red or yellow-brown liquid oozing from the dead bark, under which the colour of wood turned to blue-black. Phytophthora cactorum (Lebert and Cohn) J. Schröt., P. citricola Sawada, and P. citrophthora were found to be the causal agents [38,39,40]. Later, another pathogen, Pseudomonas syringae pv. aesculi was isolated from Aesculus hippocastanum trees, causing similar symptoms. The bacterium causes wilting, cankers, necrotic areas on the main stem and branches, rust or black coloured liquid oozing on the bark and dieback [41,42,43,44]. McEvoy et al. (2016) isolated bacterial species present in bark of diseased horse-chestnut trees. Among them, three isolates were identified as Brenneria nigrifluens [45].
Characteristic symptoms were observed on horse-chestnut trees in parks and public areas in several locations of Budapest between 2015 and 2019. Symptoms included cracks on the trunks and branches, and a sticky substance was oozing from them, which stained the bark of the trees brown. The aims of this study were to isolate and identify the pathogen that induced the previously observed symptoms.

2. Materials and Methods

In several locations of Budapest, samples were collected from symptomatic inner bark and the exudate of horse-chestnut trees (Aesculus hippocastanum). The selected trees were in parks and public areas and showed a dark brown substance oozing on their trunks and branches (Table 2). Samples were transferred to the laboratory of Department of Plant Pathology (Hungarian University of Agriculture and Life Sciences, Buda Campus, Institute of Plant Protection, Budapest) for isolation and were examined by conventional and molecular identification methods. Pure cultures were obtained on King’s medium B (KB) [46] and colonies were characterised.
Biochemical tests were performed on all horse-chestnut isolates using an API 20E Kit (BioMérieux) according to the manufacturer’s instructions. After inoculation of the test strips, they were incubated for 24 h at 26 °C. Gram testing was done with a 3% KOH solution [47]. Hypersensitive reaction was assayed on Nicotiana tabacum L. ‘Xanthi’ plants, according to the method described by Klement (1963) [48]. Tobacco leaves were inoculated with bacterial suspension (107 cfu/mL) of cultures of the three horse-chestnut strains and an Erwinia amylovora strain, as a positive control, which were grown on KB medium for 24 h. Hypersensitive response was evaluated 24–48 h after inoculation.
Pathogenicity tests were conducted with all horse-chestnut isolates on one-year-old Aesculus hippocastanum saplings. Two methods were performed with bacterial suspensions (107 cfu/mL) of 24-h-old cultures grown on KB medium. Control plants were treated with sterile distilled water. The first method was the following: the bases of leaves on the top of shoots were inoculated with the bacterial suspension using a sterile syringe with a needle. After inoculation, plants were covered with transparent plastic bags for 72 h and sprayed with water to provide humidity for the infection. Three days later, plastic bags were removed. In the second method, 1–1.5 cm long wounds were made with a sterile scalpel on the trunks of the saplings and were inoculated with the bacterial suspension. After inoculation, the wounds were sealed with Parafilm, and symptoms were monitored. Three months later, symptoms were evaluated in the case of both methods.
Conventional methods were followed for molecular-based identification. DNA was extracted from colonies of 24 h cultures and the 16S rRNA gene was amplified using the primer sets 63F/1389R [49]. The PCR reaction mixture contained (50 µL final volume) 5 µL of 10 × PCR buffer, 3 µL of 25 mM MgCl2, 2 µL of dNTP’s (5 mM), 1 µL of forward primer (20 µM) (63f–5′-CAG GCC TAA CAC ATG CAA GTC-3′), 1 µL of reverse primer (20 µM) (1389r–5′-GGG CGG WGT GTA CAA GGC-3′), 1 µL Taq DNA polymerase (5 U/µL), and 3 µL of template DNA. The amplification parameters were an initial denaturation at 95 °C for 5 min, followed by 35 cycles at 94 °C for 15 s, 55 °C for 30 s and 72 °C for 1 min 30 s. Final elongation was accomplished at 72 °C for 10 min. Gel electrophoresis was performed on 1% agarose gel (w/v) to visualize amplicons. PCR product purification was carried out using a High Pure PCR Product Purification Kit (Roche, Basel, Switzerland) following the manufacturer’s instructions. Purified PCR products were ligated into pGEM-T Easy vector and transformed into competent cells of Escherichia coli DH5α strain. Plasmid DNA was purified by Quantum Prep Plasmid Miniprep kit (Bio-Rad, Hercules, CA, USA) following the manufacturer’s protocol. Nucleotide sequences were compared to members of Pectobacteriaceae family available at the National Center for Biotechnology Information (NCBI) database using Nucleotide BLAST. Then, 16S rRNA gene sequences of the Brenneria and Lonsdalea species, originating from Hungary and other countries known to cause bark cankers and exudate oozing on various tree species, were downloaded from the GenBank sequence database. Nucleotide sequences were aligned by CLUSTAL W [50]. Determination of the best fit substitution model and maximum likelihood analysis were conducted by using MEGA version 7 [51] with the following settings: Hasegawa–Kishino–Yano model [52], bootstrap method supported by 1000 replicates, gamma distribution with invariant sites (G + I), and partial deletion of gaps/missing data treatment with a 95% cut-off. The 16S rRNA gene sequences of BpAes1, BpAes2 and BpAes3 were deposited in the NCBI GenBank (MW662111, MW662109, MW662110).

3. Results

In 2015 and since, vertical cracks were observed on the trunks and branches of Aesculus hippocastanum trees in Budapest. From these cracks and pruning wounds, brown-coloured, watery liquid has been oozing in humid and warm conditions (Figure 1). These symptoms were similar to those caused by bacteria isolated from willow (Brenneria salicis), walnut, plane (B. nigrifluens), and poplar (Lonsdalea populi) trees in Hungary [16,22,53].
All strains isolated from horse-chestnut trees grew on King’s medium B., colonies were convex, round, white, slightly bluish, and had entire margins. Biochemical characterisation of the horse-chestnut isolates was done by using an API 20E biochemical test and the results were compared with a Hungarian Brenneria nigrifluens strain isolated from walnut (Bn–WalnutZa–Hun1) and a type strain for Brenneria nigrifluens (LMG 2694T). Differences between strains in phenotypic characteristics are shown in Table 3.
All isolates were Gram-negative and a hypersensitive response was not induced on the tobacco leaves. Pathogenicity tests were evaluated three months after inoculation. In the case of the first method, shoots of horse-chestnut saplings were inoculated; around the inoculation point, small, necrotic lesions were observed, but without oozing exudate. In the case of the second method, wounds were made on the stems of horse-chestnut trees. Around these wounds, necrotic tissues were observed but brown, liquid ooze did not appear. On the control plants, no symptoms were detected. From all inoculated saplings necrotic tissues were visible, and the same bacteria were successfully reisolated.
In the PCR assay, a 1294-bp-long DNA fragment was amplified by the 63F/1389R primer pair spanning the partial 16S rRNA gene fragments of the isolates. The nucleotide sequences of the amplified PCR fragments of the16S rRNA gene of horse-chestnut isolates were determined. BLAST search (using MEGA Nucleotide BLAST) results showed that the determined sequences were 99.36% similar to Brenneria nigrifluens strains sequences (LR735272, MG950413, LN875281, LN875278) isolated in Hungary, 97.28%, 96.89%, and 96.65% similar to B. corticis (MH206610), B. roseae subsp. roseae (KY231168), and B. populi subsp. brevivirga (NR_158058), respectively. A maximum likelihood tree was created from the nucleotide sequences of the three horse-chestnut isolates, as well as from Brenneria, Lonsdalea, Dickeya, and Pectobacterium type strains (obtained from NCBI GenBank), with Cronobacter sakazakii as an outgroup (Figure 2). The isolates analysed in the present study formed a separate branch and were found to be the closest phylogenetic relationship to the Hungarian Brenneria nigrifluens strain which was described on walnut (HF936707) trees. Besides, they showed lower similarity to other B. nigrifluens strains: the type strain (AJ233415) and a strain (JX484738) which originates in Serbia.

4. Discussion

The present study contributes to the knowledge of phytopathogenic bacteria in horse-chestnut trees, being an important tree species because Aesculus hippocastanum is on the Red List of the International Union for Conservation of Nature and Natural Resources (IUCN) as a "vulnerable" species [54]. Since 2015, cracks on the bark and brown exudate oozing have been observed on Aesculus hippocastanum trees in several places, streets, parks, and public places of Budapest. The symptoms were similar to those caused by the Brenneria and Lonsdalea species isolated from other tree species in previous years [12,17,18,31,32,33,35]. However, horse chestnut was not originally known as a host plant of these pathogens. In Europe, bleeding canker disease of horse-chestnut trees is an increasing problem [43,44,55,56]. In some cases, Pseudomonas syringae pv. aesculi was isolated from symptomatic horse-chestnut trees [44], but Phytophthora species could cause similar symptoms as well [38,39,40]. McEvoy et al. isolated and identified several bacterial species from diseased Aesculus hippocastanum trees such as Brenneria nigrifluens, Pseudomonas marginalis and P. putida [45].
Based on the results presented in this study, the three horse-chestnut isolates are Gram-negative, did not induce hypersensitive response and are grouped to Brenneria nigrifluens strains and isolates (closest to a walnut isolate from Hungarian) formed a single branch on a maximum likelihood phylogenetic tree, but differences in phenotypic characteristics were observed. Further characterization of isolates could be performed as by DNA–DNA hybridisation, fatty acid analysis, and MLSA trees based on housekeeping genes (atpD, gyrB, infB, rpoB).
However, the pathogenicity tests demonstrated that the isolated strains were not able to induce cankers or bacterial oozing on the samplings, though they provoked small necrotic lesions at the inoculation point. Since the pathogenicity trials were carried out in the field and at the time of and after inoculation, the temperature and moisture content were not high enough to produce the entire infection. Probably, it could take years for the typical symptoms to develop.
On the other hand, other bacterial and non-bacterial pathogens also could have contribution to the formation and development of symptoms, but, in the present study, neither were investigated. There are many questions that are awaiting answers, e.g., What kind of impact could Brenneria species have? Could one or more other pathogens play a role in the development of symptoms? What are the long-term consequences of the infection? Is the disease present in other countries?
Four years after sampling, the diseased tree seemed alive but was in weaker condition than the horse-chestnut trees around it. Damage caused by Cameraria ohridella was visible on all of them. In the parks and streets of Budapest, plant protection of Aesculus hippocastanum—and of other ornamental trees—is a significant challenge because of the legislation in force in public areas, a lack of plant protection products, the height of the trees, and the lack of sterilisation during pruning. In some cases, plant protection experts try to control horse-chestnut leaf miners with the injection method, by making a small hole in the trunk of the tree and using insecticide. The problem with this method is that the hole created is an open door to plant pathogens—occasionally bacterial oozing was observed from these holes.

5. Conclusions

To the best of our knowledge this is the first report on Brenneria species isolated from horse-chestnut trees in Hungary.
Studies from previous years demonstrate that the number of hosts of Brenneria and Lonsdalea species is increasing in the number of countries reporting its appearance. It is important to have better knowledge of these pathogens, to inhibit their spread and find a way to protect these valuable trees. Deciduous ornamental trees play an important role in cities; they give shade in summer heat; provide proper habitat for birds, squirrels, and insects; clean the air; and, it is proven, they have stress-relieving effect. These are just a few arguments in favour of their protection.

Author Contributions

Conceptualization, I.T.-B. and A.V.; methodology, I.T.-B. and A.V.; investigation, I.T.-B., A.V., G.B. and R.G.; resources, I.T.-B., A.V., L.P. and G.Z.; data curation, I.T.-B.; writing—original draft preparation, I.T.-B.; writing—review and editing, I.T.-B., L.P. and A.V.; visualization, I.T.-B. and A.V.; funding acquisition, A.V. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Ministry for Innovation and Technology within the framework of the Thematic Excellence Programme 2020–Institutional Excellence Subprogram (TKP2020-IKA-12) for research on plant breeding and plant protection, and by grant NKFIH–Postdoctoral Excellence Programme (PD-16-121062).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article.

Acknowledgments

The authors wish to acknowledge Miklós Bozsó (PHDNRL), Ede Böszörményi and Gergő Kovács, for their technical assistance. Anita Végh was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences and by the ÚNKP-21-5-MATE-6 New National Excellence Program of the Ministry for Innovation and Technology from the source of the National Research, Development and Innovation Fund.

Conflicts of Interest

The authors declare that they have no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Forests 13 00227 g001 550
Figure 1. Cracks and brown ooze on horse-chestnut trees in Budapest. (Imola Tenorio-Baigorria, Rita Gyuris).
Figure 1. Cracks and brown ooze on horse-chestnut trees in Budapest. (Imola Tenorio-Baigorria, Rita Gyuris).
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Forests 13 00227 g002 550
Figure 2. Maximum likelihood tree based on 16S rRNA gene nucleotide sequences of the Aesculus hippocastanum strains, as well as from the Brenneria, Lonsdalea, Dickeya, and Pectobacterium species. An outgroup is included, Cronobacter sakazakii. Bar—0.01 substitutions per nucleotide position; rhombus—indicates the isolates characterized in this study.
Figure 2. Maximum likelihood tree based on 16S rRNA gene nucleotide sequences of the Aesculus hippocastanum strains, as well as from the Brenneria, Lonsdalea, Dickeya, and Pectobacterium species. An outgroup is included, Cronobacter sakazakii. Bar—0.01 substitutions per nucleotide position; rhombus—indicates the isolates characterized in this study.
Forests 13 00227 g002
Table
Table 1. Summary of Brenneria species, host plants, and sequence information.
Table 1. Summary of Brenneria species, host plants, and sequence information.
SpeciesHost Plant(s)LocationType StrainCulture Collection NumbersAccession Numbers (GenBank)Sequence Length (bp)
Brenneria salicis (syn. Erwinia salicis)Salix alba L. [13], Populus sp.,
Alnus glutinosa (L.) Gaertn. [14]		Belgium [15], Hungary [16], Italy [17], Iran [18], Japan [19], Netherlands [20], United Kingdom [13] ATCC 15712, CIP 105204, CCUG 48855, CFBP 802, DSM 30166, LMG 2698, NCPPB 447AJ233419 U80210
Z96097		1499
1455
1471
Brenneria nigrifluens (syn. Erwinia nigrifluens)Juglans regia L. [8]France [21], Hungary [22], Iran [23], Italy [24], Serbia [25], Spain [26], United States [8]EN 101ATCC 13028, CCUG 48853, CFBP 3616, CIP 105198, DSM 30175, ICMP 1578, LMG 2694, NCPPB 564AJ2334151500
FJ6118841387
U802031456
Z960951484
Brenneria rubrifaciens (syn. Erwinia rubrifaciens)Juglans regia L. [27]Spain [28], United States [27]533 CATCC 29291, DSM 4483, ICPB ER103, NCPPB 2020, PDDCC 1915AJ2334181491
FJ6118851387
Z96098 U802071484
1457
Lonsdalea quercina (syn. Erwinia quercina, Brenneria quercina, Lonsdalea quercina subsp. quercina)Quercus agrifolia Née., Quercus wislizenii A. de Candolle [29]Spain [30], United States [29] ATCC 29181, ATCC 29281, CCUG 48867, CFBP 3617, CFCC 10717, CIP 105201, DSM 4561, ICMP 1845, LMG 2724, NCPPB 1852AJ2334161509
AJ2234691531
KX5377491323
NR_0419751509
NR_1147061531
Brenneria alni (syn. Erwinia alni)Alnus glutinosa, Alnus cordata Loiseleur [9]Italy [9]PVFi20ATCC 700181, CCUG 48887, CIP 104916, DSM 11811, ICMP 12481, NCPPB 3934AJ2334091496
AJ2234681502
EU4906031224
Brenneria goodwiniiQuercus robur L. [31], Q. petraea (Mattuschka) Lieblein,United Kingdom [31], Iran [32]R-43656BCC 845, DSM 27058, FRB 141, LMG 26270, NCPPB 484KM0322711450
Q. castaneifolia von Meyer,JN5442021344
Q. macranthera Hohenacker [32]
Brenneria roseae subsp. roseaeQuercus cerris L. [33], Q. castaneifolia,United Kingdom [33], Iran [32]FRB 222FRB 222, LMG 27714, NCPPB 4581KF3082911344
Q. macranthera [32]NR_1256981344
Brenneria roseae subsp. americanaQuercus kelloggii Newb. [31]United States [31]FRB 223FRB 223, LMG 27715, NCPPB 4582KF3082921344
NR_1256991344
Brenneria populi Hauben et al. 1998aPopulus x euramericana (Dode) Guinier (unpublished)Spain NCPPB 4299JX392892
Brenneria populi subsp. brevivirgaPopulus x euramericana [34]China [34]D8-10-2-5CFCC 11935, KTC 42841KX9871351450
NR_1580581450
Brenneria populi subsp. populi (syn. Brenneria populi Li et al. 2015.)Populus x euramericana [34]China [34]D9-5CFCC 11963, KCTC 42088KJ6325181450
NR_1446041450
Brenneria corticisPopulus x euramericana [35]China [35]gBX10-1-2CFCC 11842, KCTC 42840MH2066101463
NR_1715151463
Table
Table 2. Summary of isolates with details of locations and dates of sampling.
Table 2. Summary of isolates with details of locations and dates of sampling.
StrainAccession NumberLocationDate of Collection
BpAes1MW662111Budapest, Hungary2015
BpAes2MW662109Budapest, Hungary2018
BpAes3MW662110Budapest, Hungary2019
Table
Table 3. Phenotypic characteristics of type strain and Hungarian strains of Brenneria nigrifluens and horse-chestnut isolates.
Table 3. Phenotypic characteristics of type strain and Hungarian strains of Brenneria nigrifluens and horse-chestnut isolates.
Characteristics12345
β-Galactosidase−−−−−
arginine dihydrolase−−−−−
lysine decarboxylase−−−−−
ornithine decarboxylase−−−−−
citrate utilization+−−−−
h2s production+−−−−
urease+−−−−
tryptophane deaminase−−+++
indole production−−−−−
acetoin production+++−−
gelatinase−−−−−
acid production from
D-glucose+++−−
D-mannitol−++++
Inositol+++++
D-sorbitol−+−−−
L-rhamnose−++++
D-sucrose+++++
D-melibiose−+−−−
Amygdalin−++++
L-arabinose+++++
Strains 1—Brenneria nigrifluens (Bn–WalnutZa–Hun1); 2—Brenneria nigrifluens (LMG 2694T); 3—BpAes1; 4—BpAes2; 5—BpAes3; +—positive; −—negative.
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Tenorio-Baigorria, I.; Botyánszki, G.; Gyuris, R.; Zsigó, G.; Palkovics, L.; Végh, A. Brenneria nigrifluens Isolated from Aesculus hippocastanum L. Bark in Hungary. Forests 2022, 13, 227. https://doi.org/10.3390/f13020227

AMA Style

Tenorio-Baigorria I, Botyánszki G, Gyuris R, Zsigó G, Palkovics L, Végh A. Brenneria nigrifluens Isolated from Aesculus hippocastanum L. Bark in Hungary. Forests. 2022; 13(2):227. https://doi.org/10.3390/f13020227

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Tenorio-Baigorria, Imola, Gergely Botyánszki, Rita Gyuris, György Zsigó, László Palkovics, and Anita Végh. 2022. "Brenneria nigrifluens Isolated from Aesculus hippocastanum L. Bark in Hungary" Forests 13, no. 2: 227. https://doi.org/10.3390/f13020227

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