Australasian Plant Pathology (2020) 49:209–212
https://doi.org/10.1007/s13313-020-00693-w
RESEARCH NOTE
Anthracnose pathogen of the succulent plant Echeveria
‘Perle von Nürnberg’
Jinai Yao 1 & Peng Huang 1 & Hanxin Chen 2 & Xiangyu Hou 1 & Deyi Yu 1
Received: 15 November 2019 / Accepted: 14 February 2020 / Published online: 18 February 2020
# Australasian Plant Pathology Society Inc. 2020
Abstract
Zhangzhou in Fujian province is the primary habitat for the succulent plant Echeveria ‘Perle von Nürnberg’ (Crassulaceae),
which has seriously suffered from anthracnose disease in recent years. Plants with typical disease symptoms were collected from
three planting bases in Zhangzhou, and pathogens were isolated for further identification. The pathogen was identified as
Colletotrichum destructivum by morphological observation, rDNA-ITS, Actin, and TUB2 gene sequence analysis, and patho-
genicity identification. This is the first ever report of anthracnose caused by C. destructivum on Echeveria ‘Perle von Nürnberg.’
Keywords Succulent plant . Echeveria ‘Perle von Nürnberg’ . Anthracnose . Colletotrichum destructivum
Succulents are ornamental plants with important commercial formulate the best control measures. In this study, we collected
application. They are small and fascinating and have consid- plant samples exhibiting anthracnose disease symptoms from
erable aesthetic value. They function as stress-relieving plants Echeveria ‘Perle von Nürnberg’ cultivation sites in three
in our lives and are present in thousands of households, of- towns in Zhangzhou, Fujian Province, China. A combination
fices, and public spaces. They have been referred to as “cute” of morphological and molecular identification techniques was
on the internet and are extremely popular (Wang et al. 2009). employed to determine the type of anthracnose disease path-
Echeveria ‘Perle von Nürnberg,’ also known as the purple ogen on the plants in order to develop prevention and control
pearl, is an important commercial succulent plant species that protocols for anthracnose disease in succulent plants with
is appreciated for its beautiful pink and purple coloring and these structures.
lotus shape. In June 2018, a survey of the Echeveria ‘Perle In June 2018, plants with anthracnose were collected from
von Nürnberg’ cultivation areas in Jiuhu Town, Chengxi three cultivation sites in Zhangzhou, Fujian Province, China.
Town, and Guanxun Town in Zhangzhou, Fujian Province, The sites were located in Jiuhu Town (124°29′3.73″N,
China, found that some Echeveria ‘Perle von Nürnberg’ plants 117°38′40.20″E), Chengxi Town (24°23′33.89″N, 117°35′
exhibited dark brown disease spots. These brown or dark 9.34″E), and Guanxun Town (24°19′19.26″N, 117°44′
brown small spots appeared as almost round water stains that 52.29″E) (Fig. 1a). After surface disinfection, small pieces
gradually expanded from the center, were concave in shape, of stem tissue were placed onto potato dextrose agar (PDA)
and had a slightly raised edge, and gradual rotting of the leaves medium for 5 d at 28 °C. Thirty-six strains exhibiting similar
was observed. This seriously affected the ornamental and eco- morphological characteristics were isolated. From the 36 path-
nomic value of Echeveria ‘Perle von Nürnberg.’ Anthracnose ogenic strains obtained via isolation, we randomly selected
on Echeveria ‘Perle von Nürnberg’ has never been reported. It two representative strains according to the year and cultivation
is urgent that the pathogen is accurately identified so as to site based on the classical characteristics of the colony, co-
nidia, and hyphae. The strains were renumbered FJCD-23
and FJCD-36 for monospore purification. The purified strains
* Deyi Yu were inoculated on PDA plates and placed in a light incubator
[email protected]
(Shanghai Xinmiao Instrument Manufacturing Co., Ltd.,
1 China) at 28 °C ± 0.5 °C with a light cycle of 12 L:12 D for
Fujian Key Laboratory for Monitoring and Integrated Management
of Crop Pests, Institute of Plant Protection, Fujian Academy of 6 d before colony morphological characteristics, color, and
Agricultural Sciences, Fuzhou 350013, Fujian, China hyphal growth status were observed. The conidial morpholog-
2
Zhangzhou Institute of Agricultural Science, ical characteristics were observed under a CX51 optical mi-
Zhangzhou 363005, Fujian, China croscope (Olympus, Japan). We observed 20 spores in each
210 J. Yao et al.
Fig. 1 The symptoms and
morphological characteristics of a
anthracnose disease in Echeveria
‘Perle von Nürnberg’ caused by
Colletotrichum destructivum. a
Typical symptoms on naturally
infected leaves. b Purified colony
of C. destructivum grown on
PDA. c conidia; d: appressorium;
e: acervulus; and f: symptom on
inoculated leaves (5 d); P.S: c. b. e
Scales = 50 μm
b c d e
f
Treatment 2d
Treatment 5d
CK 5d
microscopic field, and five fields were observed in total (Lin DNA of the two purified pathogen strains with a fungal
et al. 2018). We discovered that the growth rate was relatively genomic DNA extraction kit (D2300-50 T, Beijing
fast on the PDA culture medium, with developed and dense Solibao Technology Co., Ltd., China). The rDNA-ITS
aerial mycelia observed. The colonies appeared flocculated gene universal primers (ITS1/ITS4), Actin gene primers
and were grayish-brown in color (Fig. 1b). The morphological (ACT-512F/ACT-783R), and TUB2 gene primers
characteristics are as follows: conidial peduncle branched or (TUB2a / TUB2b) were used for PCR amplification
unbranched, borne at the base of conidial disk, colorless; co- (Bio-Rad, USA) of the genomic DNA of the pathogen.
nidia oblong, colorless, unicellular, smooth, obtuse at both Fragments of about 550 bp, 250 bp, and 600 bp were
ends, some with oil spheres, and 12.0–18.0 μm × 3.5– amplified. The PCR amplification products were recov-
5.0 μm in length (Fig. 1c). The appressoria are black with ered and transformed into Escherichia coli. Plasmids were
large shape differences, some irregular, some nearly round extracted, and positive clones were selected and sent for
(Fig. 1d). Conidial disks give off numerous black acicular DNA sequencing at Sangon Biotech (Shanghai, China).
setae (Fig. 1e). According to the taxonomic system of Wu After sequencing and alignment, we found that the related
and Zhang (1994) and Ma et al. (2008), the morphological gene sequences were completely identical and that these
characteristics of the aforementioned types of conidia were two representative strains were the same pathogenic fun-
similar to C. destructivum but different from other gal species. The rDNA-ITS, Actin, and TUB2 gene se-
Colletotrichum species. Therefore, the two strains were iden- quences of FJCD-23 and FJCD-36 were submitted to
tified as C. destructivum. NCBI/GenBank (www.ncbi.nlm.nih.gov/genbank/), and
To further classify the strains, we also conducted mo- accession numbers were obtained: FJCD-23 (Accession
lecular identification analyses. The method of Xu et al. No. MN151373, MN151368, MN138460) and FJCD-36
(2018) was used as a reference to extract the genomic (Accession No. MN151374, MN151369, MN138461).
Anthracnose pathogen of the succulent plant Echeveria ‘Perle von Nürnberg’ 211
We used NCBI/BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi) control. The inoculation site was then wrapped with sterile
to align the rDNA-ITS, Actin, and TUB2 gene sequences water-soaked cotton to retain moisture. Five host plants
of the two strains and found that they shared 99.45%, 98. were inoculated for every treatment, with three replicates.
84%, and 97.85% similarity with the C. destructivum Within 7 days post-inoculation (DPI), signs of disease
model of strain gene sequences (Accession No. were observable in the stems of the host plants, and the
JX625169, KC843544, GU935894), respectively. The pathogens were isolated and purified from hosts that dem-
rDNA-ITS, Actin, and TUB2 gene sequences of the onstrated classical symptoms. Morphological observations
homologous strains (Colletotrichum destructivum, and molecular identification were then carried out. The
Accession No. JX625169, KC843544, GU935894; results showed that on the second day of wounding inocu-
Colletotrichum panacicola, Accession No. GU935869, lation, the epidermis became nearly circular and water-
GU944757, GU935888; Colletotrichum higginsianum, stained, and brown or dark-brown spots appeared at the
Accession No. KF550281, MK118048, GU935892; inoculation site, following which the disease spots gradu-
Colletotrichum ocimi, Accession No. KU498272, ally expanded. At 5 DPI, the disease spots appeared as
KM105432, KM105502; Colletotrichum gloeosporioides, water stains that were concave in the center, slightly
Accession No. HQ645076, MK784769, DQ084518; uplifted at the edges, and sometimes scattered with small
Colletotrichum fuscum, Accession No. MF992186, black spots, and the leaves gradually rotted, which was
KM105389, KM105459; Colletotrichum lini, Accession very similar to the natural disease symptoms on plants
No. EU400148, MF563540, KM105520; Colletotrichum grown in the field (Fig. 1f). No disease occurred in the
linicola, Accession No. AB046609, JQ005828, Echeveria ‘Perle von Nürnberg’ plants that were inoculat-
KJ556347; Colletotrichum tabaci, Accession No. ed with the PDA agar block control. Hosts with typical
KM105206, KM105414, KM105486; Colletotrichum disease symptoms were again used for isolation and puri-
v i g n a e, A c ce s s i o n N o . K M 1 0 5 18 3 , K M 1 0 5 39 2 , fication of the pathogens, and morphological observations
K M 1 0 5 4 6 3 ; C o l l e t o t r i c h u m a m e r i c a e - b o re a l i s , and molecular identification were carried out. We found
Accession No. MF805737, KM105434, MH632716; that the re-isolated strain was similar to the inoculated
Colletotrichum lentis, Accession No. MH864629, strain. From this result, we confirmed that the pathogen
KY241670, KM105521; Colletotrichum spaethianum, of Echeveria ‘Perle von Nürnberg’ stem rot disease is
Accession No. MH453905, MH985157, GU228101; C. destructivum.
Colletotrichum guizhouensis, Accession No. JX625170, In this study, we isolated and purified an Echeveria ‘Perle
KC843538, JX625199; Colletotrichum coccodes, von Nürnberg’ anthracnose pathogen and conducted morpho-
Accession No. AJ301984, GQ856787, MH800202; logical observations, molecular identification, and pathoge-
Fusarium oxysporum, Accession No. MK962470, nicity measurements, which mutually validated each other.
MH511658, MH888083) were used to construct a We confirmed that this disease is caused by C. destructivum
polygenic associative phylogenetic tree. It was found that infection. This is the first ever report that anthracnose disease
the FJCD-23 and FJCD-36 strains clustered along the in Echeveria ‘Perle von Nürnberg’ is caused by
same branch with C. destructivum. According to these C. destructivum. The plant pathogen C. destructivum has a
results, we determined that the FJCD-23 and FJCD-36 wide range of hosts. Leaf damage by insects or pruning, which
strains were C. destructivum. cause physical injury sites, allows it to invade the leaf tissue
One-year-old healthy plants with flat leaves and trauma- via natural orifices. The disease rapidly expands to cause plant
free stems were obtained from the Jiuhu Town cultivation death under suitable conditions. The anthracnose pathogen
site and brought back to the laboratory along with the cul- C. destructivum is the primary disease agent of Echeveria
tivation bag. Sterile water was used to gently wash the ‘Perle von Nürnberg’ in Zhangzhou, Fujian, China.
stems and leaves of Echeveria ‘Perle von Nürnberg.’ Moreover, it has the tendency to spread to other succulents,
Following recovery, the plants were used for pathogenicity which affects the cultivation and product quality of the succu-
measurements. The purified FJCD-23 and FJCD-36 path- lents. There is thus a need for further confirmation of the
ogenic strains were selected for pathogenicity measure- pathogenesis of this pathogen and an analysis of its pathoge-
ments according to the method of Tambe et al. (2017). nicity patterns and characteristics in order to formulate effec-
Wounding inoculation was employed, whereby three tive prevention and control protocols.soli.
healthy leaves were randomly selected from the lower part
of the plant, and the upper epidermis of the leaves was Acknowledgments The authors would like to thank the Program of
Fujian Provincial Science and Technology Department (grant number
punctured with a sterile needle. The hyphal block of
2017 N0063, 2018R1025-2, 2019R1024-5) and Projects of Fujian
0.5 × 0.5 cm to be tested was applied to the wounded area Academy of Agricultural Sciences (grant number STIT2017-2-2,
of the leaves, and the sterile PDA agar block was used as a DEC201907), which funded the research.
212 J. Yao et al.
Compliance with ethical standards This article follows the Tambe E, Afanga YA, Bechem ET (2017) Morphological and molecular
experimental guidelines of the country. identification of fungi associated with corm rot and blight symptoms
on plantain (Musa paradisiaca) in macro-propagators. Int J Biol Sci
11:2793–2808
Conflict of interest The authors declare that they have no conflict of
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screening and utilization in Xiamen. Subtropical Plant Sci 38:69–73
Wu WP, Zhang ZM (1994) Colletotrichum (Colletotrichum Cda.) II tax-
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