Research Article |
Corresponding author: Wei He ( hewei@bjfu.edu.cn ) Corresponding author: Ying Zhang ( yzhang@bjfu.edu.cn ) Academic editor: Huzefa Raja
© 2019 Lin Zhao, Jing Cai, Wei He, Ying Zhang.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Zhao L, Cai J, He W, Zhang Y (2019) Macrophomina vaccinii sp. nov. causing blueberry stem blight in China. MycoKeys 55: 1-14. https://doi.org/10.3897/mycokeys.55.35015
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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.
Vaccinium, stem blight, Botryosphaericeae, taxonomy, pathogenicity
Blueberries (Vaccinium spp.) are popular fruits because of their health benefits health, such as enhancing brain memory and preventing heart disease (
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 (
The genus Macrophomina was introduced based on M. phaseolina, and assigned in the Botryosphaeriaceae (Botryosphaeriales) (
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.
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 (
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) (
GenBank accession numbers of isolates included in this study (newly generated sequences are in bold).
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 (
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
Three isolates of Macrophomina vaccinii (
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.
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.
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,
from “Vaccinium”, in reference to the host genus.
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;
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.
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,
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)) (
All the three isolates of Macrophomina vaccinii (
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.
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 |
|
12.63 ± 7.32 a |
Macrophomina vaccinii |
|
12.38 ± 0.48 a |
Macrophomina vaccinii |
|
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 | – |
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 (
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) (
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.
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 (
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).