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Macrophomina vaccinii sp. nov. causing blueberry stem blight in China
expand article infoLin Zhao, Jing Cai, Wei He, Ying Zhang
‡ Beijing Forestry University, Beijing, China
Open Access

Abstract

Blueberries (Vaccinium spp.) have been widely cultivated in China because of their nutritional benefits and economic value. Blueberry stem blight has become one of the most severe diseases influencing blueberry productivity and quality in China. In this study, eight fungal isolates were obtained from twenty stem blight lesions of blueberry collected in Nanping, Fujian province, China. Asexual stage was observed after inducing sporulation, the morphology of which agrees with Macrophomina in the black, smooth, hard sclerotia and ellipsoid to obovoid, smooth hyaline conidia with apical sheath. Furthermore, DNA sequences of concatenated ITS, tef1-α, TUB, and ACT loci indicated that these isolates belong to a novel fungal species. The distinguishing morphological characteristics, such as the wider conidia and larger conidiomata pycnidial, also support its new status. Thus a novel fungus, Macrophomina vaccinii, was described in this study. Pathogenicity tests indicated that M. vaccinii could cause stem blight of blueberry.

Keywords

Vaccinium, stem blight, Botryosphaericeae, taxonomy, pathogenicity

Introduction

Blueberries (Vaccinium spp.) are popular fruits because of their health benefits health, such as enhancing brain memory and preventing heart disease (Shi and Liu 2009, Popović et al. 2018). Blueberries have been commercially cultivated worldwide, particularly in the USA, Canada and a few European countries (Evans and Ballen 2014). Blueberry cultivation in China started in 1981, and the planted area has reached 31,210 hectares with total production of 114, 905 t in 2017 (Li et al. 2018). Blueberries have been widely cultivated mainly in Guizhou, Shandong and Liaoning province (Xu et al. 2015, Li et al. 2018). Stem blight disease was one of the most prevalent diseases in blueberry cultivation areas in China, which has seriously affected the growth of blueberry plants, fruit quality and productivity (Yu et al. 2012, 2013a, b, Xu et al. 2015, Xu 2016).

A number of fungal species have been reported causing stem blight, dieback or stem canker of blueberries. For instance, Botryosphaeria dothidea, Lasiodiplodia theobromae, Neofusicoccum ribis, and N. parvum caused stem blight of highbush or rabbiteyes blueberries in USA (Milholland 1972, Creswell and Milholland 1988, Smith 2004, Wright and Harmon 2009, 2010, Koike et al. 2014). Macrophomina phaseolina (Tassi) Goid caused stem blight of highbush blueberries in Serbia (Popović et al. 2018). Neofusicoccum parvum caused stem blight and dieback of highbush blueberries in Mexico (Boyzo-Marin et al. 2016). Diaporthe ambigua, D. australafricana, D. neotheicola, D. passiflorae, Pestalotiopsis clavispora, P. neglecta, and Truncatella angustata caused stem canker and dieback of highbush blueberries in Chile (Espinoza et al. 2008, Elfar et al. 2013), and Godronia cassandrae caused stem dieback of highbush blueberry in Norway (Stromeng and Stensvand 2011).

The genus Macrophomina was introduced based on M. phaseolina, and assigned in the Botryosphaeriaceae (Botryosphaeriales) (Crous et al. 2006, Phillips et al. 2013). Thus far, three species are accommodated within Macrophomina, viz. M. phaseolina, M. pseudophaseolina Crous, Sarr & Ndiaye, and M. euphorbiicola A.R. Machado, D.J. Soares & O.L. Pereira (Phillips et al. 2013, Sarr et al. 2014, Machado et al. 2019). Macrophomina phaseolina is a soil- or seed-borne polyphagous pathogen, causing charcoal rot disease on about 500 plant species of more than 100 families throughout the world (Su et al. 2001, Babu et al. 2007, Sarr et al. 2014). In Serbia, M. phaseolina was reported as a causal agent causing foliage death and brown discoloration of internal vascular stem tissues of highbush blueberry in 2015 (Popović et al. 2018). So far, M. pseudophaseolina has been reported causing charcoal rot disease on six plant species, viz. Abelmoschus esculentus, Arachis hypogaea, Hibiscus sabdariffa, Vigna unguiculata, Gossypium hirsutum, Ricinus communis, and associated with seed decay of Jatropha curcas (Sarr et al. 2014, Machado et al. 2019). Macrophomia euphorbiicola has only been reported as the causal agent of the charcoal rot on Ricinus communis and Jatropha gossypiifolia (Machado et al. 2019).

In the course of an ongoing survey of biodiversity of fungi causing stem blight of blueberries in China, a new taxon with general characteristics of Macrophomina was collected. The aim of this study was to identify the new isolates based on morphological characteristics and multigene phylogenetic analysis, and determine their pathogenicity on the blueberry.

Materials and methods

Sample collection, fungal isolation and morphological studies

This study was conducted at the Blueberry Production Garden in the suburb area of Nanping, Fujian province, China. Twenty diseased or dead stems (about 30 cm in length) were collected from blueberry branches in February, 2018. Wood segments (0.5 × 0.5 × 0.2 cm) cut from the diseased lesion boundary or dead tissue were surface sterilized (Pavlic et al. 2004) and incubated on malt extract agar (MEA, 2%) for fungal strains. Petri-dishes were incubated in the dark at 28 °C until fungal colonies were observed. Pure cultures were obtained by hyphal tips from the margin of the suspected Macrophomina colonies, which were subcultured on fresh MEA and maintained at 28 °C.

To induce sporulation of conidia, isolates were cultivated on synthetic nutrient-poor agar (SNA) with autoclaved pine needles placed onto the medium, and incubated at 25 °C under near-UV light (mainly 340 nm) (Dou et al. 2017b). Pycnidia produced on the pine needles were morphologically described and characterized following the protocol of Dou et al. (2017a, b). Measurements of conidia, conidiogenous cells and microconidia were made from water mounts. Measurements and digital photographs were made using a Nikon Coolpix 995 digital camera connected to a trinocular Leitz Orthoplan microscope and processed with Adobe Photoshop Elements 10 software. Fungal isolates and specimens were deposited at Beijing Forestry University (BJFU) with duplicates in the China General Microbiological Culture Collection Center (CGMCC) and the Mycological Herbarium of the Institute of Microbiology, Chinese Academy of Sciences (HMAS) (Table 1).

GenBank accession numbers of isolates included in this study (newly generated sequences are in bold).

Species Sample number GenBank accession number
ITS tef1-a TUB ACT
Botryosphaeria dothidea CBS 115476 AY236949 AY236898 AY236927
CBS 110302 AY259092 AY573218 EU673106
Macrophomina euphorbiicola CMM 4045 KU058928 KU058898 MF457657 MF457654
CMM 4134 KU058936 KU058906 MF457658 MF457655
CMM 4145 KU058937 KU058907 MF457659 MF457656
M. phaseolina CBS 162.25 KF531826 KF951996 KF531805 KF951803
CBS 227.33 KF531825 KF952000 KF531806 KF951807
CPC 21388 KF951703 KF952074 KF952165 KF951843
CPC 21392 KF951705 KF952076 KF952167 KF951844
CPC 21395 KF951706 KF952077 KF952168 KF951846
CPC 21399 KF951707 KF952078 KF952169 KF951847
CPC 21443 KF951734 KF952104 KF952194 KF951872
CPC 21444 KF951735 KF952105 KF952195 KF951873
CPC 21445 KF951736 KF952106 KF952196 KF951874
M. pseudophaseolina CPC 21394 KF951786 KF952148 KF952228 KF951913
CPC 21402 KF951789 KF952151 KF952231 KF951916
CPC 21403 KF951790 KF952152 KF952232 KF951917
CPC 21417 KF951791 KF952153 KF952233 KF951918
CPC 21459 KF951794 KF952156 KF952236 KF951921
CPC 21501 KF951796 KF952158 KF952238 KF951923
CPC 21524 KF951799 KF952161 KF952240 KF951925
CPC 21527 KF951801 KF952163 KF952242 KF951927
CPC 21528 KF951802 KF952164 KF952243 KF951928
M. vaccinii CGMCC 3.19503 MK687450 MK687426 MK687434 MK687442
CGMCC 3.19504 MK687451 MK687427 MK687435 MK687443
CGMCC 3.19505 MK687452 MK687428 MK687436 MK687444
CGMCC 3.19506 MK687453 MK687429 MK687437 MK687445
CGMCC 3.19507 MK687454 MK687430 MK687438 MK687446
CGMCC 3.19508 MK687455 MK687431 MK687439 MK687447
CGMCC 3.19509 MK687456 MK687432 MK687440 MK687448
CGMCC 3.19510 MK687457 MK687433 MK687441 MK687449

DNA extraction, PCR amplification and sequencing

DNA was extracted from mycelia grown on MEA plates with CTAB plant genome DNA fast extraction kit (Aidlab Biotechnologies Co., Ltd, Beijing, China). The internal transcribed spacer of rDNA (ITS) was amplified and sequenced with primers ITS-1 and ITS-4 (White et al. 1990). The translation elongation factor-1α (tef1-α) was amplified and sequenced with primers EF1-688F and EF1-1251R (Alves et al. 2008). The β-tubulin gene (TUB) was amplified and sequenced with primers Bt2a and Bt2b (Glass and Donaldson 1995). The actin gene (ACT) was amplified and sequenced with primers ACT-512F and ACT-2RD (Carbone and Kohn 1999, Sarr et al. 2014). PCR amplification and sequencing followed the protocols of Zhang et al. (2009).

Sequence alignment and phylogenetic analysis

DNA sequences of concatenated ITS, tef1-α, TUB, and ACT loci were analyzed to investigate the phylogenetic relationships among Macrophomina species with DNA sequences available from GenBank (http://www.ncbi.nlm.nih.gov/genbank/), as well as the sequences generated herein (Table 1). A multiple alignment was conducted with MEGA v. 6 (Tamura et al. 2013) and analyses were performed in PAUP V. 4.0b10 (Swofford 2002). Prior to phylogenetic analysis, ambiguous sequences at the start and the end were deleted and gaps manually adjusted to optimize the alignment. Maximum parsimony (MP) was conducted with heuristic searches as implemented in PAUP with the default options method (Zhang et al. 2008). Analyses with gaps treated as missing data were conducted under different parameters of maximum parsimonious criteria as outlined in Zhang et al. (2008). Clade stability was evaluated in a bootstrap analysis with 1,000 replicates, random sequence additions with the maxtrees set to 1,000 and other default parameters as implemented in PAUP. Maximum likelihood (ML) was also conducted using heuristic searches with the default options method as implemented in PAUP. For the ML analysis, best-fit model of nucleotide evolution (HKY+G) was selected by hierarchical likelihood ratio test (hLRT) in MrModeltest 2.3 (Posada and Crandall 2001). A bootstrap analysis with 1,000 replicates was used to test the statistical support of the branches. Trees were viewed in TreeView 1.6.6 (Page 1996). The nucleotide sequences reported in this paper were deposited in GenBank. Trees and alignments were deposited in TreeBase (https://treebase.org/treebase-web/home.html, submission ID: 24410).

Pathogenicity test

Three isolates of Macrophomina vaccinii (CGMCC 3.19503, CGMCC 3.19505, and CGMCC 3.19510) obtained in this study were used to conduct a pathogenicity test. The pathogenicity test was performed on 2-year blueberry stems (cv. O’Neal) in a humid chamber at 28 °C with semi-shaded conditions. Stems for inoculation were surface sterilized with 75% ethanol for 1 min before making a tangential cut (5 mm in length) on the bark (Espinoza et al. 2009). A 5-mm-diameter MEA medium with mycelial was taken from the 3-day colony, which was placed on to the wounded site, and subsequently covered with parafilm. Three replicates were conducted for each isolate. Noncolonized MEA agar plugs were used as negative controls. Pathogenicity was determined by the length of the necrotic lesion caused by the tested isolates three weeks after inoculation. Fungal isolates were re-isolated from the infected tissue, and morphological characterization and DNA sequence comparisons were conducted to fulfill Koch’s postulates. Mean comparisons were conducted using Tukey’s Honest Significant Difference test (HSD, α = 0.05) in R (Version 3.2.2, R Inc. Auckland, NZL).

Results

Phylogeny

Phylogenetic analysis of the concatenated ITS, tef1-α, TUB and ACT sequence dataset comprising 1,426 bp revealed 129 parsimony-informative characters. The outgroup taxon was Botryosphaeria dothidea. The heuristic search with random addition of taxa (1,000 replicates) generated 5,000 most parsimonious trees of 141 steps (CI = 0.972, RI = 0.990, RC = 0.962, HI = 0.028). In both analyses (MP and ML), M. phaseolina and M. vaccinii formed a well-supported clade (MP BS = 99%, ML BS = 91%). Macrophomina pseudophaseolina and M. euphorbiicola formed another clade which lacks of bootstrap support (MP BS = 68%, ML BS = 67%, Fig. 1).

Figure 1. 

Maximum parsimony tree generated from sequence analysis of the concatenated ITS, tef1-α, TUB and ACT dataset. Designated out group taxa is B. dothidea. Maximum parsimony (MP) and maximum likelihood (ML) bootstrap support greater than or equal to 60% are shown above the nodes (* = value less than 60%). The positions of the Macrophomina vaccinii isolates are indicated in bold and red text.

Taxonomy

Macrophomina vaccinii Y. Zhang ter & L. Zhao, sp. nov.

MycoBank No: 830282
Figure 2

Holotype

CHINA, Fujian province, Nanping city, Jianyang district, Huilong village, from blighted stem of southern high bush (Vaccinium corymbosum × V. darrowii), 26 Feb. 2018, L. Zhao (HMAS 255479): ex-type living culture, CGMCC 3.19503.

Etymology

from “Vaccinium”, in reference to the host genus.

Description

Sexual stage not observed. Asexual stage: Sclerotia developing on SNA, black, smooth, hard, 40–100 µm diam. Conidiomata pycnidial, dark brown to black, solitary or gregarious, up to 400 µm diam., each opening by a central ostiole. Conidiogenous cells lining the inner surface of the conidioma, hyaline, subcylindrical, each proliferating several times percurrently near the apex, 9–16 × 3–4 µm, young conidiogenous cells each covered by a mucous layer that extends over the apex of the developing conidium. Conidia ellipsoid to obovoid, smooth, (18–)20–29(–33) × (8–)9–11(–12) µm (av. 24.8 × 10.1 µm, n = 60, L/W ratio = 2.5, range from 2.3 to 2.8), immature conidia hyaline, enclosed in a mucous sheath, that upon dehiscence encloses the top half of the conidium, transformed into two lateral tentaculiform, apical mucoid appendages (type C; Nag Raj 1993), no pigmented conidia observed after 30 days incubation. Microconidia aseptate, hyaline, smooth, guttulate to granular, straight to curved, ellipsoid to subcylindrical to irregular, 5–9(–10) × 3–5 µm.

Figure 2. 

Macrophomina vaccinii (from ex-type: CGMCC 3.19503). a Pycnidia forming on pine needle b Sclerotia on the synthetic nutrient-poor agar c Conidiogenous cells d Microconidia e–f Conidia with apical appendages (arrows). Scale bars: 1 mm (a); 10 µm (b–f).

Culture characteristics

Colonies on MEA at 25 °C in darkness, with even margins, sparse aerial mycelia. On MEA buff, turning pale olivaceous to olivaceous-black with dense, black sclerotial masses. Colonies reaching 58.6 mm on MEA after 2 d in the dark at 25 °C.

Additional specimens examined

CHINA, Fujian province, Nanping city, Jianyang district, Huilong village, from blighted stem of southern high bush (Vaccinium corymbosum × V. darrowii), 26 February 2018, L. Zhao (Paratype, HMAS 255480): living culture, CGMCC 3.19505; (HMAS 255481): living culture, CGMCC 3.19510.

Note

Based on phylogenetic analysis, M. vaccinii and M. phaseolina formed a well-supported clade. Morphologically, the wider conidia of Macrophomina vaccinii can be distinguishable from M. phaseolina ((8–)9–11(–12) µm (av. 10.1 µm) vs. (6–)8(–9) µm (av. 8 µm)) (Sarr et al. 2014). In addition, the larger-sized pycnidia of M. vaccinii are also distinguishable from M. phaseolina (up to 400 µm diam. vs. up to 300 µm diam.) (Sarr et al. 2014). A comparison of the 264 nucleotides across the tef1-α gene region of M. vaccinii and M. phaseolina (CBS 227.33) reveals 5 base pair differences (1.9%) (Table 3).

Pathogenicity test

All the three isolates of Macrophomina vaccinii (CGMCC 3.19503, CGMCC 3.19505, and CGMCC 3.19510) were pathogenic on the blueberry stems. Brown lesions appeared on the inoculated spots after 3 days of inoculation for mycelia (Fig. 3). The diseased spots turned brown and lesion area enlarged after 7 days inoculation (Fig. 3). After inoculation for 3 weeks, the length of necrotic lesion reached up to 20 cm, and the infected xylem tissue turned light-brown (Fig. 3). The wounded area of the inoculated stems was the one that was most significantly higher than those of the control groups, while no significant difference was detected among these three inoculated treatments (Fig. 3, Table 2).

Koch’s postulates were performed by successful pathogen re-isolation from all the necrotic stems. The morphology and DNA sequences of these new isolates were consistent with the initial inoculate.

Figure 3. 

Macrophomina vaccinii causes stem blight of blueberry. a Death of the blueberry (Vaccinium spp.) plants in the field b Symptoms of stem blight of blueberry in the field c Symptoms of Macrophomina vaccinii after three days inoculation d Symptoms of Macrophomina vaccinii after one-week inoculation e Symptoms of Macrophomina vaccinii after three weeks inoculation f Symptoms of blueberry twig of Macrophomina vaccinii after three weeks inoculation.

Pathogenicity on 2-year blueberry stems (cv. O’Neal) using mycelia of Macrophomina vaccinii after 3 weeks.

Species Isolate Blueberry stems inoculated with Mycelia ± SD (cm)
Macrophomina vaccinii CGMCC 3.19503 12.63 ± 7.32 a
Macrophomina vaccinii CGMCC 3.19505 12.38 ± 0.48 a
Macrophomina vaccinii CGMCC 3.19510 10.75 ± 2.87 a
Noninoculated control 0.00 ± 0.00 b

Major tef1-a and TUB and ACT base pair differences of Macrophomina vaccinii, M. phaseolina, M. pseudophaseolina and M. euphorbiicola.

Species Base pair difference Position of nucleotides difference
tef1-a TUB ACT
M. vaccinii and M. phaseolina G instead of A 11
C instead of T 41
C instead of G 48
A instead of C 75
A instead of G 160
T instead of C 76
M. vaccinii and M. pseudophaseolina A instead of G 10, 24
C instead of T 27, 31, 48, 103, 186 280, 313
G instead of A 101, 144, 208 119, 192
A instead of T 142
T instead of C 145, 197, 217, 227, 247 56 76, 192
T instead of A 219
C instead of A 202
M. vaccinii and M. euphorbiicola C instead of T 14, 23, 33, 193, 221 280, 313
A instead of G 24
T instead of C 43, 250 56 76, 192
C instead of G 48
C instead of A 106 202
G instead of A 144, 211 119, 192 83
A instead of C 185
G instead of C 200

Discussion

Macrophomina is a cosmopolitan genus, with a broad host range and colonizing more than 500 crops and non-crop species, such as soybean, common bean, corn, sorghum, cowpea, peanut and cotton (Su et al. 2001, Ndiaye et al. 2010, Sarr et al. 2014, Sun et al. 2015). In this study, Macrophomina vaccinii was collected from the lesion of stem blight in Fujian province in China, a subtropical area in China. Macrophomina phaseolina, the most common species of Macrophomina, is considered as economically more important in subtropical and tropical countries with semi-arid climates, which tends to occur in hot and dry conditions (Wrather et al. 1997, 2001, Smith and Wyllie 1999, Radwan et al. 2014). Charcoal rot of beans is caused by M. phaseolina, however, this has frequently been reported in the northern part of China, with a disease incidence of 80% in Beijing and Tianjin (Zhang et al. 2009, 2011, Sun et al. 2015).

So far, seven species have been assigned within Macrophomina, viz. M. euphorbiicola, M. limbalis, M. phaseoli, M. phaseolina, M. philippinensis, M. pseudeverniae and M. pseudophaseolina. However, M. limbalis was transferred to Dothiorella (as D. limbalis), M. pseudeverniae to Didymocyrtis (as D. pseudeverniae), while M. phaseoli and M. philippinensis were treated as the synonym of M. phaseolina. Thus, only three species, viz. M. euphorbiicola, M. phaseolina and M. pseudophaseolina are currently accommodated within Macrophomina. Morphologically, wider conidia of M. vaccinii ((8–)9–11(–12) µm) are distinguishable from M. phaseolina ((6–)8(–9) µm) and M. pseudophaseolina ((7.5–)8(–9) µm) (Sarr et al. 2014). The larger-sized pycnidia of M. vaccinii (up to 400 µm diam.) can also be distinguishable from M. phaseolina (up to 300 µm diam.) and M. pseudophaseolina (up to 300 µm diam.) (Sarr et al. 2014). In addition, the smaller-sized sclerotia of M. vaccinii (40–100 µm diam.) also differs from M. phaseolina (100–400 µm diam.) and M. pseudophaseolina (100–400 µm diam.) (Sarr et al. 2014). Macrophomina euphorbiicola lacks morphological descriptions, and only DNA sequences are available for species comparison (Machado et al. 2019).

Phylogeny based on concatenated ITS, tef1-α, TUB and ACT DNA sequences indicated that the subclade comprising eight isolates of Macrophomina vaccinii are closely related to M. phaseolina (Fig. 1). A comparison of the tef1-α regions DNA sequence data of M. vaccinii and M. phaseolina revealed a 1.9% base pair difference. A comparison of the 266 nucleotides across the tef1-α gene region of M. vaccinii and M. pseudophaseolina (CPC 21417) reveals 17 base pair differences (6.39%). Although the morphological characteristics of M. euphorbiicola cannot be obtained, a comparison of the 269 nucleotides across the tef1-α gene region between M. vaccinii and M. euphorbiicola (CMM 4134) shows 13 base pair differences (4.83%) (Table 3). Following the recommendations of Jeewon and Hyde (2016) and Tennakoon et al. (2018), there is sufficient evidence to justify our taxon as a new species.

Pathogenicity tests conducted on 2-year blueberry stems (cv. O’Neal) indicated that inoculation of Macrophomina vaccinii were pathogenic on blueberry stems which causes the stem turn brown with necrotic lesions. Similar symptoms caused by M. phaseolina have been reported on blueberry in Serbia, resulting in foliage death, and brown discoloration of internal vascular tissues at the basal part of the bush (Popović et al. 2018). The brown lesion caused by M. vaccinii and M. phaseolina on blueberries differs from the widely reported charcoal rot diseases caused by Macrophomina phaseolina and M. pseudophaseolina (Su et al. 2001, Salik 2007, Yang et al. 2005, Zhang et al. 2011, Sarr et al. 2014, Sun et al. 2015).

Acknowledgements

This work was supported by the National Natural Science Foundation of China (General Program, 31770015, 31370063), NSFC Projects of International Cooperation and Exchanges (3155461143028) and National Science and Technology Foundation Project (2014FY210400).

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