Verticillium Wilt Caused by Verticillium dahliae and V. nonalfalfae in Potato in Northern China

Abstract

Potato (Solanum tuberosum L.) is one of the most important staple foods in many parts of the world including China. In recent years, Verticillium wilt has become a severe threat to potato production in China. During 2015 to 2016, 287 samples of symptomatic potato plants were collected from 15 counties in five provinces from northern China. One hundred and eighty-seven Verticillium-like colonies were isolated from these samples and identified to species based on cultural and morphological characteristics, and multigene phylogeny based on the partial sequences of actin (ACT), elongation factor 1-alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GPD), and tryptophan synthase (TS) genes. A consensus-rooted most parsimonious phylogenetic tree was generated from the data. One hundred and fifteen isolates comprising 61.5% of the total were identified as Verticillium dahliae, and the remaining 38.5% of the isolates were identified as V. nonalfalfae. V. dahliae was widely distributed in Shaanxi (84.1%), Inner Mongolia (76.7%), Gansu (12.8%), and Qinghai (100%, representing a single isolate). V. dahliae was not recovered from the samples in Ningxia. V. nonalfalfae dominated the collections from Gansu (87.2%) and Ningxia (100%) but was also recovered from Shaanxi (15.9%) and Inner Mongolia (23.3%) at lower frequencies. Neither V. albo-atrum nor V. alfalfae was recovered from the sampled areas. The V. nonalfalfae isolates were predominantly isolated from the samples collected from altitudes above 1,800 m, and in contrast, V. dahliae isolates were mainly recovered from fields sampled below 1,800 m. The optimum temperature for the colony growth of V. nonalfalfae was lower (20°C) than that for V. dahliae (25°C). Pathogenicity tests demonstrated that V. dahliae and V. nonalfalfae were both pathogens of potato Verticillium wilt, with V. dahliae isolates exhibiting higher virulence than V. nonalfalfae isolates regardless of the collection area of the species. This is the first documentation of V. nonalfalfae infecting S. tuberosum in China and the higher altitudes associated with infections of V. nonalfalfae anywhere in the world.

Full text

Verticillium Wilt Caused by Verticillium dahliae and V. nonalfalfae
in Potato in Northern China
Rui Jing, Haiyuan Li, Xiaoping Hu, and Wenjing Shang,
†
State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant
Protection, Northwest A&F University, Yangling, Shaanxi 712100, China; Ruiqing Shen and Chengjin Guo, Plant Protection Institute, Ning-
xia Agriculture and Forestry Academy, Yinchuan, Ningxia 750002, China; Qingyun Guo, Key Laboratory of Agricultural Integrated Pest
Management In Qinghai Province, Academy of Agriculture and Forestry Science, Qinghai University, Xining, Qinghai 810016, China; and
Krishna V. Subbarao, Department of Plant Pathology, University of California, Davis, Salinas, CA 93905
Abstract
Potato (Solanum tuberosum L.) is one of the most important staple
foods in many parts of the world including China. In recent years, Ver-
ticillium wilt has become a severe threat to potato production in China.
During 2015 to 2016, 287 samples of symptomatic potato plants were
collected from 15 counties in five provinces from northern China.
One hundred and eighty-seven Verticillium-like colonies were isolated
from these samples and identified to species based on cultural and mor-
phological characteristics, and multigene phylogeny based on the par-
tial sequences of actin (ACT), elongation factor 1-alpha (EF1a),
glyceraldehyde-3-phosphate dehydrogenase (GPD), and tryptophan
synthase (TS) genes. A consensus-rooted most parsimonious phyloge-
netic tree was generated from the data. One hundred and fifteen isolates
comprising 61.5% of the total were identified as Verticillium dahliae,
and the remaining 38.5% of the isolates were identified as V. nonalfal-
fae.V. dahliae was widely distributed in Shaanxi (84.1%), Inner Mon-
golia (76.7%), Gansu (12.8%), and Qinghai (100%, representing a
single isolate). V. dahliae was not recovered from the samples in Ning-
xia. V. nonalfalfae dominated the collections from Gansu (87.2%) and
Ningxia (100%) but was also recovered from Shaanxi (15.9%) and In-
ner Mongolia (23.3%) at lower frequencies. Neither V. albo-atrum nor
V. alfalfae was recovered from the sampled areas. The V. nonalfalfae
isolates were predominantly isolated from the samples collected from
altitudes above 1,800 m, and in contrast, V. dahliae isolates were
mainly recovered from fields sampled below 1,800 m. The optimum
temperature for the colony growth of V. nonalfalfae was lower
(20°C) than that for V. dahliae (25°C). Pathogenicity tests demon-
strated that V. dahliae and V. nonalfalfae were both pathogens of potato
Verticillium wilt, with V. dahliae isolates exhibiting higher virulence
than V. nonalfalfae isolates regardless of the collection area of the
species. This is the first documentation of V. nonalfalfae infecting
S.tuberosum in China and the higher altitudes associated with infections
of V. nonalfalfae anywhere in the world.
Potato (Solanum tuberosum L.), a member of the Solanaceae fam-
ily, is fourth among the staple food crops in the world following
wheat, rice, and corn (Desjardins et al. 1995). More than a billion
people worldwide consume potato, and the annual global production
exceeds 300 million metric tons (https://cipotato.org/crops/potato/).
China and India accounted for nearly one third of the world’s potato
yield and since 2009, potato production in China has surpassed all
nations (Jia et al. 2011; Li and Yang 2016). Verticillium wilt is a
common disease and a major threat to potato production throughout
the world (Powelson and Rowe 1993; Rowe et al. 1987; Rowe and
Powelson 2002). The pathogen colonizes xylem elements following
successful infection and interferes with water uptake, leading to foliar
discoloration, necrosis, and wilt (Rowe and Powelson 2002). Yield
losses caused by Verticillium wilt typically range from 10 to 15%
but can reach up to 50% in severely affected fields (Johnson et al.
1986; Powelson and Rowe 1993).
Verticillium dahliae and V. albo-atrum were previously consid-
ered the causal agents of Verticillium wilt of potato (Pegg and
Brady 2002; Rowe and Powelson 2002), but recent reassessment
of the taxonomy of the genus Verticillium showed that V. albo-atrum
was not the major pathogen causing Verticillium wilt of potato
(Inderbitzin et al. 2011a). The V. albo-atrum species reported in
previous literature was likely what is currently taxonomically re-
ferredtoasV. nonalfalfae. It has a limited host range, and relies
on the melanized hyphae to survive in soil. Verticillium wilt caused
by V. albo-atrum is distributed in cooler climatic regions, where av-
erage temperatures do not exceed 21°C during the growing season
(Powelson and Rowe 2008; Rowe et al. 1987). V. tricorpus,
V. nigrescens (now named Gibellulopsis nigrescens), and V. nubilum
can also infect potato and other plants, but they are considered as
weak pathogens or soil saprotrophs (Barbara and Clewes 2003;
Isaac 1949; Pegg and Brady 2002; Slattery and Eide 1980). In con-
trast, V. dahliae causes vascular wilt diseases on more than 200
plant species worldwide (Pegg and Brady 2002). It can survive as
microsclerotia in field soil for 10 years or more (Menzies and Griebel
1967). Thus, once a field is infested by V. dahliae, the disease
becomes a significant constraint in production. Verticillium wilt of
potato was typically uncommon in China, but it was recently ob-
served in Gansu Province and the causal agent was determined to
be V. dahliae (Chen et al. 2013).
The taxonomy of the genus Verticillium has a long and compli-
cated history. The genus used to contain about 190 species (Zare
et al. 2004), which were distinguished by the resting structure mor-
phology, conidia size, conidiophore size and pigmentation, number
of phialides per whorl, and the formation of yellow-pigmented hy-
phae (Inderbitzin et al. 2011a). However, with the advent of molec-
ular systematics, most of the distantly related species were removed
from the genus Verticillium, including plant pathogenic species such
as V. nigrescens and V. theobromae (now named as Musicillium the-
obromae) (Zare et al. 2007), which reduced the number of species
within the genus Verticillium to five (Barbara and Clewes 2003). Us-
ing multigene phylogeny, in a landmark study, Inderbitzin et al.
(2011a) added five new species to this genus (Barbara and Clewes
2003), including V. zaregamsianum,V. isaacii,V. klebahnii,V.alfal-
fae,andV. nonalfalfae (Inderbitzin et al. 2011a). However, pathogens
†
Corresponding author: W. Shang; E-mail: shangwj@nwsuaf.edu.cn.
Funding: The study was sponsored by the National Natural Science Founda-
tion of China (31371888) and partially by The Open Foundation of the Key
Laboratory of Agricultural Integrated Pest Management, Qinghai University
(2017-ZJ-Y16) and by the Pilot Project of Scientific and Technical Innova-
tion, Ningxia Academy of Agriculture and Forestry Sciences (NKYJ‐15‐33).
*The e-Xtra logo stands for “electronic extra”and indicates that one sup-
plementary table is published online.
Accepted for publication 7 April 2018.
©2018 The American Phytopathological Society
1958 Plant Disea se / Vol. 102 No. 10
Plant Disease •2018 •102:1958-1964 •https://doi.org/10.1094/PDIS-01-18-0162-RE

causing Verticillium wilt of potato have so far not been investigated
systematically.
Owing to the importance of potato crop in China and the emerging
Verticillium wilt that threatens its production, it is important to char-
acterize the causal agents and develop disease management methods
to sustain production. The objectives of this study were therefore to
characterize isolates of Verticillium obtained from symptomatic po-
tato plants from production fields in northern China and to determine
the distribution of the different species causing Verticillium wilt as
influenced by the ecozones of potato production in northern China.
Materials and Methods
Sample collection and pathogen isolation. From 2015 to 2016,
26 potato fields were sampled from 15 counties of Gansu, Ningxia,
Shaanxi, Inner Mongolia, and Qinghai provinces during potato flow-
ering, when symptoms were easily observed. The number of sur-
veyed fields depended on the Verticillium wilt incidence in the
potato fields in each province. Four fields were sampled from Gansu,
four from Ningxia, nine from Shaanxi, eight from Inner Mongolia,
and one field from Qinghai (Table 1). Individual fields varied in size,
ranging from one-tenth of a hectare to one-half hectare as most
growers in China are small land holders. Plants showing characteris-
tic Verticillium wilt symptoms were sampled from severely diseased
fields. The number of plants sampled per field depended on observed
disease severity with 11 to 25 plants sampled from severely symp-
tomatic fields (>25% of plants showed symptoms) and 3 to 10 plants
sampled from lesser symptomatic fields (<25% of plants showed
symptoms). Additional information regarding the sampled fields is
listed in Table 1. Disease incidence within each field was assessed
by walking the fields in an X pattern and the disease status recorded
for 20 plants along each of the four transects. Stems of random symp-
tomatic plants ranging from 3 to 25 were collected from each location
where disease severity was assessed and returned to the laboratory
for pathogen isolation.
Symptomatic stems were cut into 1- or 2-cm pieces, surface disin-
fested with 75% alcohol for 5 to 10 s, rinsed with sterile distilled wa-
ter three times, air-dried on sterile filter paper for 3 min, and then
plated onto potato dextrose agar (PDA) amended with 100 mgml
−1
streptomycin sulfate. The plates were incubated at 25 ± 1°C in the
dark. Verticillium-like colonies that grew from plated stems were
transferred to fresh PDA plates, single-spored, and stored at –80°C
as conidial suspensions diluted with glycerol (25%) for later use.
Morphological characterization and colony growth of isolates.
The identification of Verticillium was initially based on morpholog-
ical characteristics as outlined by Inderbitzin et al. (2011a). All iso-
lates were examined for colony morphology and pigmentation on
Table 1. Verticillium wilt disease incidence and Verticillium species isolated from potato stem samples collected from commercial production fields across north-
ern China
Province, Field Altitude (m) Cultivar Disease incidence (%)
Number of
samples
Number of
V. dahliae
Number of
V. nonalfalfae
Gansu 64 5 34
Lijiabaozhen, Andingqu 1,939 Unknown 56 9 5 0
Xiangquanzhen, Andingqu 2,063 Shepody 74 21 0 13
Huichuanzhen, Weiyuan 2,270 Unknown 60 19 0 15
Gaofengxiang, Andingqu 2,565 Unknown 65 15 0 6
Ningxia 22 0 11
Zhonghexiang, Yuanzhouqu 1,802 Zhuangshu 3 8 10 0 7
Malianxiang, Xiji 1,895 Qingshu 9 5 5 0 2
Xitanxiang, Xiji 1,978 Qingshu 9 4 4 0 2
Xinyingxiang, Xiji 2,077 Unknown 1 3 0 0
Shaanxi 101 53 10
Yuyangqu 1,200 Shepody 1 5 1 1
Yangqiaopanzhen, Jingbian 1,230 1106-7 1 4 2 1
Muhuguanzhen, Shangzhouqu 1,254 Unknown 1 11 1 0
Sidahaocun, Bainijingzhen,
Dingbian
1,355 Unknown 0 3 0 0
Zhangluokengcun,
Bainijingzhen, Dingbian
1,370 Zihuabai 4 7 3 0
Shuanghaizicun, Bainijingzhen,
Dingbian
1,390 Unknown 70 18 16 0
Yanggaocun, Dongkengzhen,
Jingbian
1,500 Zihuabai 50 12 2 6
Caoyaoxiancun, Dongkengzhen,
Jingbian
1,500 Zihuabai,
Jizhangshu 12
18 16 10 0
Wangzequzhen, Jingbian 1,510 Zihuabai, Shepody 61 25 18 2
Inner Mongolia 97 56 17
Hongqinghezhen, Ejin Horo
Banner
1,320 Unknown 85 24 19 0
Subuergazhen, Ejin Horo Banner 1,420 Kexin 1 45 8 6 0
Jiningqu, Ulanqab 1,402 Unknown 1 5 0 2
Sanchakouxiang, Chahar Right
Front Banner
1,450 Favorita 1 7 1 2
Bayinxilezhen, Zhuozi 1,580 Favorita 70 23 16 5
Sanshengtaicun, Kezhen,
Wuchuan
1,600 Kexin 1 30 13 6 5
Daluerhaocun, Kezhen,
Wuchaun
1,680 Shepody 55 11 6 2
Hongpanxiang, Chahar Middle
Front Banner
1,700 Kexin 1 2 6 2 1
Qinghai 31 0
Taizixiang, Huzhu 2,535 Unknown 5 3 1 0
Total 287 115 72
Plant Disease /October 2018 1959

PDA, morphological features including mycelia, conidia, verticil-
lately arranged phialidic conidiophores, and resting structure were
also recorded for each isolate by examining the cultures using a
compound microscope (Olympus BX51, Japan). Colony morphol-
ogy was captured using a Canon 80D camera. The width and length
of 80 conidia from five isolates representing each species were mea-
sured using a compound microscope. These data identified two spe-
cies of Verticillium from among the isolates.
To determine the response of the two species to temperature, five
isolates of each species were incubated at 15, 20, 25, and 30°C on
complete medium (CM) (dextrose 10 g, peptone 2 g, yeast extract
1 g, acid hydrolyzed complex proteins 1 g, NaNO
3
6 g, KCl 0.5 g,
MgSO
4
·7H
2
O 1 g, KH
2
PO
4
1.5 g, ager 15 g, water 1 liter). A 5-
mm-diameter agar disk containing mycelial growth of each isolate
was obtained from the edge of 2-week-old colonies of each isolate
and transferred to the center of five plates containing CM. The plates
were incubated at each temperature and the diameters of colonies
were measured at 2-day intervals until the growth reached the edge
of the plates in any one treatment. The experiment was repeated once.
DNA extraction and PCR amplification. The conidial suspen-
sions from each isolate were spread on PDA plates covered in ad-
vance with cellophane membrane and mycelia were collected 5 days
later. DNA was extracted from each isolate using CTAB method
described by Zolan and Pukkila (1986). Four protein coding genes
employed (Inderbitzin et al. 2011a) in our study included partial se-
quences of actin (ACT), glyceraldehyde-3-phosphate dehydrogenase
(GPD), elongation factor 1-alpha (EF1a), tryptophan synthase (TS),
and the primer pairs for polymerase chain reaction (PCR) amplification
employed were: VActF/VActR for ACT, VEFf/VEFr for EF1a,
VGPDf2/VGPDr for GPD, and VTs3f/VTs3r for TS. Additional de-
tails regarding primer sequences, amplicon length, and annealing tem-
perature followed were as described in Inderbitzin et al. (2011b). All
primers were synthesized by Invitrogen (Shanghai, China). PCR was
run with EasyTaq DNA Polymerase provided by TransGen Biotech
company (Beijing, China) in a 25 ml reaction volume, and the PCR
procedure consisted of an initial denaturation step for 5 min at 94°C,
then 35 cycles of denaturation at 94°C for 30 s, annealing for 30 s at
the related primer pair, extension for 1 min at 72°C, and final extension
at 72°C for 10 min. The PCR products were detected in a 1% agarose
gel electrophoresis and were photographed with UV transilluminator
(AlphaImager EP, U.S.A.), and all of the correct PCR products were
sequenced with the corresponding forward primers by Invitrogen
(Shanghai, China). The resulting sequences were compared by Ba-
sic Local Alignment Search Tool (BLAST) against the National
Center for Biotechnology Information (NCBI) database, and the
Verticillium sequences with greater than 99% sequence homology
were downloaded for phylogenetic analyses (Supplementary Table
S1). The sequence data for two representative strains of each Verticil-
lium species were obtained from GenBank and used in the phyloge-
netic analyses.
Phylogenetic analysis. All single-gene sequences were aligned
using CLUSTALX version 1.83 with the default settings and edited
by BioEdit to eliminate the ambiguously aligned positions and diver-
gent regions prior to phylogenetic analyses. The combined four-locus
dataset for all isolates were analyzed using maximum parsimony in
Molecular Evolutionary Genetics Analysis (MEGA) 6 with the de-
fault settings, and bootstrap supports were based on 1,000 replicates.
Gibellulopsis nigrescens sequence from GenBank was chosen as an
outgroup. A partition homogeneity test was performed in PAUP*
version 4.0b10 using 1,000 random repetitions to determine whether
the ACT,GPD,EF, and TS sequence data could be combined. Tree
length, consistency index (CI), retention index (RI), and rescaled
consistency index (RC) were calculated using MEGA6.
Pathogenicity test. Pathogenicity tests of five V. nonalfalfae iso-
lates from each province and five V. dahliae isolates were performed
on potato (Table 2). Since the status of V. dahliae as a pathogen of
potato is well established, only five isolates were tested in contrast
to higher number of isolates of V. nonalfalfae. Potato tubers (cv. At-
lantic) obtained from Gansu Ailan Potato Seed Company (Dingxi,
China) were planted into 15-cm-diameter pots filled with pasteurized
Table 2. Mean disease severity and relative marginal effects for Verticillium wilt severity on potato caused by V. nonalfalfae and V. dahliae isolates
Species, Isolates Source
Disease severity
a
Relative marginal effects
b
Mean Estimate 95% CI
V. nonalfalfae
WLCBJNQ201 Chahar Middle Front Banner 3.0 0.39 (0.24–0.57)
WC137 Wuchuan 3.4 0.50 (0.32–0.69)
WC37 Wuchuan 3.0 0.40 (0.22–0.62)
ZZ015 Zhuozi 3.8 0.64 (0.47–0.77)
ZZ018 Zhuozi 3.6 0.62 (0.44–0.77)
DXWY059 Weiyuan 3.5 0.56 (0.42–0.69)
DXADQ037 Andingqu 2.3 0.15 (0.08–0.27)
DXWY076 Weiyuan 3.3 0.47 (0.35–0.59)
DXADQ033 Andingqu 3.1 0.43 (0.29–0.58)
DXADQ043 Andingqu 3.4 0.52 (0.35–0.69)
JB87 Jingbian 3.0 0.38 (0.27–0.51)
JB058 Jingbian 3.0 0.39 (0.24–0.57)
JB183 Jingbian 3.0 0.38 (0.27–0.51)
JB066 Jingbian 3.1 0.42 (0.32–0.52)
JB192 Jingbian 2.8 0.32 (0.16–0.54)
GYZH027 Yuanzhouqu 3.4 0.51 (0.38–0.65)
GYXJ022 Xiji 3.0 0.40 (0.22–0.62)
GYXJ021 Xiji 3.0 0.40 (0.22–0.62)
GYZH024 Yuanzhouqu 3.9 0.66 (0.47–0.81)
GYZH003 Yuanzhouqu 3.3 0.47 (0.27–0.68)
V. dahliae
DXADQ054 Andingqu 4.1 0.73 (0.54–0.86)
DB083 Dingbian 4.0 0.69 (0.48–0.84)
YQ204 Ejin Horo Banner 3.8 0.64 (0.47–0.77)
WC225 Wuchuan 3.9 0.68 (0.53–0.80)
JB187 Jingbian 4.1 0.73 (0.54–0.86)
a
Disease severity was assessed on a scale of 1 to 5: 1 = no disease symptoms, 2 = slight wilting and discoloration of lower leaves, 3 = moderate wilting involving
less than 1/2 of the leaves on the plants, 4 = severe wilting involving more than 1/2 of the leaves on the plants, 5 = plant dead from wilt (Hunter et al. 1968).
b
Relative marginal effect and 95% CI were calculated from analysis of the rank values of disease severity.
1960 Plant Disea se / Vol. 102 No. 10

sand/potting soil mixture (2:1, v/v). Plants were grown in a controlled
environment greenhouse (25 ± 3°C with a photoperiod of 14 h) for
4 weeks. Spore suspensions from each isolate cultivated on PDA
for 7 days were prepared by adding sterile distilled water and scrap-
ing the cultures with a sterile spatula. The concentration of conidia in
the suspension from each isolate was adjusted to approximately 10
7
conidia/ml (Vallad et al. 2006) using a hemacytometer. Eight 4-
week-old potato plants were inoculated by flooding the soil around
the roots with 30 ml spore suspensions for each isolate, and eight
seedlings were inoculated with 30 ml sterile deionized water as a con-
trol. Plants were arranged in a completely random design on green-
house benches. Inoculated plants were monitored for Verticillium
wilt symptom development relative to the uninoculated control
plants weekly. Disease severity was recorded on each plant at 7 weeks
postinoculation using a scale of 1 to 5 in which: 1 = no disease symp-
toms, 2 = slight wilting and discoloration of lower leaves, 3 = moderate
Fig. 1. Most parsimonious rooted tree based on the ACT,EF,GPD,andTS sequences of isolates of Ver ti ci lli um species inferred from maximum parsimony analysis using
MEGA 6. Gibellulopsis nigrescens as outgroup. PD identifiers in bold are representative isolates of the different Verticillium species. DXADQ =Andingqu; DXWY =Weiyuan;
GYZH =Yuanzhouqu; GYXJ =Xiji; JB =Jingbian; DB =Dingbian; YLYYQ =Yuyangqu; ZZ =Zhuozi; WLCBJNQ =Jiningqu; WC =Wuchuan; WLCBYQQ =Chahar Right
Front Banner; WLCBYZQ =Chahar Middle Front Banner.
Plant Disease / October 2018 1961

wilting involving less than 1/2 of the leaves on the plants, 4 = severe
wilting involving more than 1/2 of the leaves on the plants, 5 = plant
dead from wilt(Hunter et al. 1968). The fungi were reisolated from dis-
eased plants as described above. The plates were incubated at 20 ± 1°C
or 25 ± 1°C in the dark and the number of plants that were developed
symptoms for either V. nonalfalfae or V. dahliae, respectively, was
recorded after 7 days incubation.
Data analysis. The mean colony diameter and standard deviation
were calculated from five V. dahliae isolates and five V. nonalfalfae
isolates with five plates for each isolate. The disease severity caused
by V. dahliae and V. nonalfalfae was analyzed by the analysis of var-
iance type statistic of ranked data using the PROC Mixed procedure
in SAS version 8.0, and relative marginal effects (RME) was gener-
ated using the procedure of LD_CI macro and the 95% confidence
intervals were constructed (Ghosh 2003; Shah and Madden 2004).
Results
Wilt incidence and isolation of Verticillium spp. from different
provinces in China. Verticillium wilt incidence on potato from 26
fields was variable between and within the provinces of Gansu (four
fields), Ningxia (four fields), Shaanxi (nine fields), Inner Mongolia
(eight fields), and Qinghai (one field), China. In Gansu, the incidence
of Verticillium wilt in four fields was >50%. In contrast, the incidence
in Ningxia and Qinghai fields was less than 10%. The incidence varied
greatly from 0 to 70% in the fields in Shaanxi, and from 1 to 85% in the
fields in Inner Mongolia (Table 1). In Shaanxi, the incidence in three
out of nine fields was >50%, and the incidence in five fields was
<5%. In Inner Mongolia, the incidence in three out of eight fields
was >50%, and the incidence in three fields was <5% (Table 1).
One hundred and eighty-seven Verticillium isolates were recov-
ered from 287 symptomatic potato stem samples. Of the total recov-
ered isolates, 39.0, 33.7, 20.9, 5.9, and 0.5% were recovered from
Inner Mongolia, Shaanxi, Gansu, Ningxia, and Qinghai, respec-
tively. One hundred and fifteen isolates identified as V. dahliae
accounted for 61.5% of all isolates; the remaining 72 isolates were
V. nonalfalfae (38.5%). V. dahliae accounted for 84.1, 76.7, and
12.8% of the total in Shaanxi, Inner Mongolia, and Gansu, respec-
tively. Only one V. dahliae isolate was recovered from Qinghai
and no V. dahliae isolates were recovered from Ningxia. V. nonalfal-
fae accounted for 100, 87.2, 23.3, and 15.9% of the total in Ningxia,
Gansu, Inner Mongolia, and Shaanxi, respectively. The high propor-
tion of V. nonalfalfae isolates were recovered from fields located at
altitudes above 1,800 m areas; for example, 87.2 and 100% of the to-
tal isolates were V. nonalfalfae in Gansu and Ningxia, respectively.
However, V. dahliae isolates were mainly collected from the fields
located at altitudes below 1,800 m in Shaanxi (84.1%) and Inner
Mongolia (76.7%) (Table 1).
Identification of the strains. Based on characteristics of resting
structures, all isolates were classified into two distinct categories with
the melanized microsclerotia or brown pigmented hyphae. The first
category consisted of 115 Verticillium isolates, and all isolates only
formed melanized microsclerotia and no other resting structures were
detected. These characteristics matched earlier descriptions for
V. dahliae (Inderbitzin et al. 2011a). The second category consisted
of 72 isolates. which only formed thick-walled and brown resting my-
celia. These characteristics matched earlier descriptions for V. albo-
atrum,V. alfalfae,andV. nonalfalfae (Inderbitzin et al. 2011a).
A consensus-rooted most parsimonious phylogenetic tree with a
multigene phylogenetic analysis generated using gene sequences of
ACT,EF1a,GPD, and TS, provided the most parsimonious tree
showing two major clades of the Verticillium species (length =
779, CI = 0.81, RI = 0.94, RC = 0.76, HI = 0.17). The representative
isolates, DXADQ049 and DXADQ055, were grouped within the
V. dahliae clade (100% bootstrap support). Since the results of the
morphological identification had indicated that isolates in the first
category were V. dahliae, only two representative isolates from first
category were tested to validate the results from morphological iden-
tification. Isolates in the second category that only formed the resting
mycelia were grouped into the clade of V. nonalfalfae (95% bootstrap
support) (Fig. 1).
Response of V. dahliae and V. nonalfafae to temperature. The
maximum growth of V. dahliae isolates occurred at 25°C, whereas
for V. nonalfalfae, it occurred at 20°C. All isolates within each spe-
cies responded similarly to temperature. Colony diameters of V. dah-
liae isolates increased with increasing temperature up to 25°C and
then declined. In contrast, colony diameters of V. nonalfalfae isolates
increased with increasing temperatures up to 20°C and then declined,
with growth completely arrested at 30°C (Fig. 2).
Pathogenicity test. All 20 isolates of V. nonalfalfae and the five
isolates of V. dahliae tested caused symptoms of Verticillium wilt
of potato. The initial wilt symptoms began as yellowing of the fo-
liage. Uneven chlorosis was observed on the lower leaves, and the
extension of the discoloration was delimited by the veins. Wilted
leaves curled upward with uneven necrosis of leaves that progressed
acropetally from the stem base (Figs. 3A and C). Finally, the entire
plant became necrotic and wilted with the dead plants remaining
erect. The vasculature of infected plants was discolored 7 weeks after
inoculation (Figs. 3D and F). Symptoms observed in greenhouse in-
oculated plants were identical to the wilt symptoms observed in the
fields. No distinction between the vascular discoloration caused by
V. dahliae and V. nonalfalfae could be made. V. dahliae and V. non-
alfalfae were reisolated from infected plants and were confirmed to
be identical to the inoculated strains based on the morphological
characteristics.
The relative virulence of the two species on potato was signifi-
cantly different with V. dahliae isolates exhibiting greater virulence
than V. nonalfalfae. Chlorosis of lower leaves caused by V. dahliae
occurred at 17 days postinoculation, while potato plants inoculated
with V. nonalfalfae showed symptoms at 21 days postinoculation.
The V. nonalfalfae isolates from each province caused Verticillium
wilt on potato. Disease severity caused by these isolates varied from
2.3 to 3.9 on a 1 to 5 point scale (Table 2). In contrast, disease sever-
ity caused by each of the V. dahliae isolates was higher than that
caused by V. nonalfalfae and varied from 3.8 to 4.1 (Table 2).
Discussion
Verticillium wilt is a major economic disease and causes signifi-
cant yield losses in all potato production regions (Powelson and
Rowe 1993; Rowe and Powelson 2002). Even though China is the
largest potato producer in the world, the status of Verticillium wilt
Fig. 2. Effect of temperature on the colony diameters of V. dahliae and V. nonalfalfae
isolates. The average colony diameter from five isolates each from V. dahliae (YQ204,
DXADQ054, WC225, DB083, and JB187) and V. nonalfalfae isolates (DXADQ043,
WC37, ZZ018, GYZH027, and JB192) with the corresponding standard deviation
are shown after 15 days of incubation. All cultures were grown on complete
medium (CM).
1962 Plant Disea se / Vol. 102 No. 10

in potato is still uncertain except for the recent report of V. dahliae
causing the disease in Gansu Province (Chen et al. 2013). As demon-
strated by this study, Verticillium wilt is widespread in the potato
production fields that were sampled across all provinces even though
the relative severity of the disease was different among the provinces.
In Gansu Province, the Verticillium wilt was most severe in all sur-
veyed fields, but in Ningxia Province, the disease was least severe.
The severity of disease varied greatly among the fields in Shaanxi
and Inner Mongolia. While this study provides a snapshot of the cur-
rent status of the disease, as observed in other systems (Atallah et al.
2011), once introduced, the pathogen not only establishes itself in in-
dividual fields but rapidly spreads from these foci to new fields with
the planting of infested seeds in new fields and/or movement of soil
and equipment between fields. Thus, the disease is likely to emerge
as a major threat to potato production in China.
Both V. dahliae and V. nonalfalfae were demonstrated to be causal
agents of Verticillium wilt on potatoes in China but their relative im-
portance differed among the different provinces where potato pro-
duction occurs. V. dahliae was the dominant species in Shaanxi
and Inner Mongolia, whereas V. nonalfalfae was the predominant
species causing the disease in Ningxia and Gansu. Previously,
V. dahliae and V. albo-atrum were the known causes of Verticillium
wilt elsewhere in the world (Pegg and Brady 2002; Rowe and Powel-
son 2002). Neither V. albo-atrum nor V. alfalfae, another species
split-off from V. albo-atrum, was found in any fields evaluated in this
study. This is the first report of V. nonalfalfae as a pathogen of potato
or any other crop in China.
Climate (altitude and the associated temperature) appear to play an
important role in the distribution of the two species causing Verticil-
lium wilt in potato. Previously, V. dahliae was the predominant cause
of Verticillium wilt on potato in production areas where average
daily summer temperatures commonly exceeded 27°C. In contrast,
V. albo-atrum was the predominant cause of Verticillium wilt in pro-
duction areas where average daily temperatures did not reach 21°C
(Powelson and Rowe 2008; Rowe et al. 1987). Similarly, we found
that V. dahliae was distributed production fields located primarily
at altitudes below 1,800 m. For example, in Shaanxi Province,
84.1% of the total isolates collected at altitudes between 1,200 and
1,510 m were V. dahliae. Similarly, in Inner Mongolia, 76.7% of
the total isolates collected at altitudes between 1,320 to 1,700 m were
V. dahliae. However, V. nonalfalfae was the primary causal agent in
fields located above 1,800 m. For example, V. nonalfalfae (87.2%)
was collected primarily at altitudes between 1,939 and 2,565 m in
Gansu Province. Similarly, V. nonalfalfae (100%) was collected at
altitudes between 1,802 to 2,077 m in Ningxia. In these two prov-
inces and at these altitudes, the average daily temperatures do not
exceed 20°C during the potato growing season (Guo et al. 2006;
Yao et al. 2008). This is further supported by the optimum tempera-
ture for colony growth of V. nonalfalfae being lower (20°C) than that
for V. dahliae (25°C). We therefore posit that temperature plays an
Fig. 3. Foliar and root symptoms of Verticillium wilt on potato seedlings caused by V. dahliae and V. nonalfalfae compared with the uninoculated control. A, D, symptom caused by
V. nonalfalfae GYZH024 strain at 7 weeks postinoculation; C, F, the symptom caused by V. dahliae DXADQ054 strain at 7 weeks postinoculation; B, E, the control plants with
no disease symptoms.
Plant Disease / October 2018 1963

important role in the distribution of the two species causing Verticil-
lium wilt in China. The above results regarding the response of the
two species to temperature are consistent with the results of Rowe
et al. (1987), even though their study compared V. dahliae and
V. albo-atrum. The significantly novel results from this study are
the association of the lower temperature requirement of V. nonalfalfae
with the higher altitudes of the potato fields from which the majority of
these isolates were collected and the association of the higher temper-
ature requirement of V. dahliae with the lower altitudes of the potato
fields from which the majority of these isolates were collected. To-
gether, the temperature and altitude may determinethe relative survival
of the two species indirectly and this needs further study.
The taxonomy of the genus Verticillium has significantly changed
since thesespecies were first reported as causing disease on potato. Ini-
tially, V. albo-atrum was reported as the pathogen of Verticillium wilt
on potato (Reinke and Berthold 1879). V. dahliae was subsequently
identified from dahlia (Klebahn 1913), and then both V. dahliae and
V. albo-atrum were determined to be the primary pathogens on potato
(Krikun and Orion 1979). V. albo-atrum in turn was divided into two
clear subspecific groups based on host specificity (L, comprising the
host adapted isolates from lucerne, and NL, comprising isolates from
all other hosts) (Heale and Isaac 1963), and the division was also
strongly supported by studies involving several molecular markers
(Morton et al. 1995; Okoli et al. 1993). A further study subdivided
V. albo-atrum into V. albo-atrum group 1 and V. albo-atrum group 2
by sequencing of the rRNA ITS regions (Robb et al. 1993). The majority
of the V. albo-atrum group 2 isolates came from potato (or soil from
fields where potatoes had been grown) from eastern Canada, Maine
(U.S.A.), and the Netherlands (Europe) (Mahuku and Platt 2002; Robb
et al. 1993). With the establishment of a new taxonomic framework for
Verticillium,V. alfalfae and V. nonalfalfae were described as the two
new species from V. albo-atrum (Inderbitzin et al. 2011a), and the three
species are morphologically indistinguishable. Inderbitzin et al. (2011a)
speculated that the name ‘V. albo-atrum’has possibly been applied more
frequently to ‘V. albo-atrum’group 1 now comprising V. alfalfae and
V. nonalfalfae,thanto‘V. albo-atrum’group 2, which is now retained
as V. albo-atrum. Regardless of the taxonomy, the current study provides
an appraisal of the importance of Verticillium wilt in potato production in
China and the pathogens that cause the disease and their relative distribu-
tion. Neither V. alfalfae nor V. albo-atrum was isolated from potato sam-
ples in China. Because of the similarity of the temperature requirements
between V. nonalfafae and V. albo-atrum, we can speculate that the path-
ogen in the previous reports in all likelihood indeed was V. nonalfalfae.
V. nonalfalfae has been reported as the cause of vascular wilt on
hops, Solanaceous crops, spinach, and forest and shade trees in Can-
ada, Japan, Cuba, Slovenia, the United Kingdom, and the United
States (Hiemstra 1998; Inderbitzin et al. 2011a; Pegg and Brady
2002; Schall and Davis 2009). Based on the results from this study,
we add potato, another Solanaceous crop, to the list of hosts of
V. nonalfalfae. The relative agricultural importance of this new spe-
cies and its host range among the crops remains to be determined.
Acknowledgments
We would like to express our sincere thanks to Hongsheng Shang, Zhouquan
Wei, Aichang Chen, Ruiqing Shen, Chengjin Guo, and Yaodong Wen, who helped
collect the samples from various locations in China.
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