Hao Jianxiu,Wang Min,Zhang Yimin,Zhou Hongyou

(College of Horticulture and Plant Protection of Inner Mongolia Agricultural University,Hohhot 010019)

Abstract:Verticillium wilt is one of the most common and widespread diseases in most potato regions of the world.It is a soil-borne and seed-borne vascular disease,which makes it difficult to control.This paper summarized the research progress of the disease,including the distribution,damage,symptoms,the morphology characteristics of the main pathogenic species,and the control measures in order to provide theoretical guidance for further research on Verticillium wilt of potato.

Key words:Potato;Verticillium wilt;Induced resistance;Pathogens;Control measures

Verticillium wilt of potato caused by Verticillium dahlia Kleb is a severe economic problem in arid and semiarid regions of the world.It’s a soil-borne hemi-biotrophic pathogen,causes wilt in more than 200 dicotyledonous plant species and is considered the primary causal agent of the potato early dying (PED) syndrome(Rowe &Powelson 2002;Johnson &Dung 2010).The symptoms of Verticillium wilt include chlorosis and necrosis,starting in the lower leaves,as well as vascular discoloration,stunting,and wilting of the infected plant (Pegg &Brady 2002).Ultimately,V.dahliae produces resting structures (microsclerotia) in the necrotic areas of infected tissues,which can sustain germination viability for as long as 10 years or more in soil (Klosterman et al.2009).When exposed to root exudate,the microsclerotia germinate and the mycelia penetrate the root epidermic cells,and then produce infectious hyphae to colonize the roots (Klosterman et al.2009).Yield losses in potato crops may reach 50%,but are more commonly in the range of 10-15% (Powelson ML &Rowe RC.1993;Rowe RC et al.1987;Rowe RC &Powelson ML.2002),whereas in lettuce,losses can easily reach 100% (Subbarao KV et al.1997).

1 Distribution and Damage of Verticillium Wilt in Potato

1.1 Distribution and Damage of Verticillium Wilt in Potato in the World

In 1879,the German Reinke and Berthold discovered and reported the disease from the potato plant firstly,and named it Verticillium wilt.The pathogen was named Verticillium alboatrum Reinke &Berthold (HARVEYCS 1965).The disease was found sporadically in Minnesota in US in 1916.From 1963 to 1965,76 percent of potato plots in the United States had Verticillium wilt to some extent,with the yield reduction of 5 to 12t per hectare.Since then,the disease has been reported all over the world and has become a worldwide disease (WANG Lili et al 2017).

1.2 Distribution and Damage of Verticillium Wilt in Potato in China

In 1944,Verticillium wilt of potato was first found in Sichuan provience in China (Peng.X.W.2003).This disease was found in Shangyi in which the disease incidence was 1.9%-20.2%,Zhangbei and Kangbei in Hebei province by Tian S.Y.(1984) (Tian.S.Y.1984) from 1972 to 1973,and the pathogen was Verticillium dahlia.And then the second pathogen reported was V.albo-atrum(Tian.S.M.1995).Another pathogen reported is in Bei Jing,Hei Longjiang and Hebei provinces was V.nigrescens Pethybr.(now renamed as Gibellulopsis nigrescens (Pethybr.) Zare) (Chen.J.D.1982).V.dahlia was found in died potato seeds in Shanxi province.The losses in potato crops may reach 50%,but are more common in the range of 20—30% which was reported in Guizhou(Zhang.L.C.2004).Because of Verticillium wilt caused by V.dahlia.verticillium wilt was found in Xinjiang and the pathogens were V.albo-atrum and V.dahlia by Wang L.L.et al.who studied the biological characteristics of these two pathogens and the screening of effective bacteriostatic agents in laboratory (Wang.L.L.et al.2011,2014a,2014b).Verticillium wilt caused by V.dahlia was reported in Gansu province (Chen.A.C.2014).Liu B.Q.reported that Verticillium wilt which can be caused by V.dahlia occurred in Inner Mongolia and Shandong provinces and study the screening of effective bacteriostatic agents in laboratory (Liu.B.Q.2016).Li S.Z.et.al.(2018) did the study of the occurrence of potato Verticillium wilt and analysis of pathogenicity differentiation of the major pathogen in seven provinces of China including Inner Mongolia,Yunnan province and so all (Li.S.Z.2018).So far,about 11 provinces,autonomous regions or municipalities in China have reported the distribution of Verticillium wilt in potatoes,and its harm degree is varied,even reduce production by more than half(Fig.1).

2 Identification of Verticillium Wilt Symptoms in Potato

The symptom of verticillium wilt which can occur in the whole growth period of potato.The symptom of the disease is different in different growth periods.The pathogen can infect leaf,stolon,underground stem and tuber and so on.It is a typical vascular disease.At first,the leaves on the lower part of the plant began to show symptoms,and gradually spread upward.Yellow patches appeared on the margins of the diseased leaves and among the main veins.Later,the color of the diseased leaves gradually became darker and brownish,and the edges of the diseased leaves were curled upward.But,the mesophyll of the single main vein and its vicinity remains green like watermelon rind.Suffered torrential rain or soaked in water after a long drought,the disease can appear acute symptoms that rhizome shriveled,leaves suddenly drooping,petioles and rhizome vascular brown.The disease plant exposed to sun is quickly parched.when the air humidity is high,the dead disease stem epidermis can be covered by a layer of gray and white mold (Dai.L.Y.1944).

3 Pathogens Caused Verticillium Wilt of Potato

3.1 The History of Verticillium Taxonomy

Nees von Essenbeck erected the genus Verticillium in 1816(Isaac I.1967;Pegg GF &Brady BL 2002) based on its unique,branched conidiophores,which form whorls capped with flaskshaped and pointed phialides carrying terminal conidia.Although a few species of the genus Verticillium have been associated with ascomycetous teleomorphs (Pegg GF &Brady BL 2002;Zare R et al.2007),V.dahliae,V.albo-atrum,and V.tricorpus are solely anamorphic with no evidence of sexual recombination or a meiosporic stage.In 1879,Reinke and Berthold firstly described wilt on potato (Solanum tuberosum),and named the causal agent V.albo-atrum (Isaac I 1967;Pegg GF &Brady BL 2002;Robinson DB et al.1957).It was not until 1913 that a second,morphologically distinct species causing wilt on dahlia (Asteraceae family) was described by Klebahn,and named V.dahliae (Isaac I.1967;Pegg GF &Brady BL 2002;Robinson DB et al.1957;Barbara DJ &Clewes E 2003).Verticillium spp.By convention,are identified based on the type of resting structures produced,namely:pigmented resting mycelium,pigmented microsclerotia,and chlamydospores.The two most distinctive features separating V.dahliae and V.albo-atrum are:(a) the production of melanized microsclerotia as survival structures by V.dahliae,in contrast to V.albo-atrum,which produces melanized hyphae but no microsclerotia (Isaac I.1967;Pegg GF &Brady BL 2002;Robinson DB et al.1957;Barbara DJ &Clewes E 2003),and (b) although V.albo-atrum fails to grow in culture or wilt plants at 30℃,V.dahliae grows and infects unhindered at 30℃(Isaac I.1967;Pegg GF&Brady BL 2002;Robinson DB et al.1957).Even though this information is written in the literature,the taxonomic debate relative to the distinctiveness of these two species continued until the late 1970s when V.dahliae was universally accepted as a species separate from V.albo-atrum (Pegg GF &Brady BL 2002;Fradin EF &Thomma BPHJ 2006).Subsequent phylogenetic studies have clearly identified V.albo-atrum and V.dahliaeas distinct taxa(Barbara DJ &Clewes E 2003;Atallah ZK et al.2007;Clewes E et al.2008;Qin QM et al.2006).

Based on host specificity,two clear sub-groups in V.alboatrum are recognized.Strains from alfalfa cause severe symptoms on this host and also on numerous other hosts,and strains from hosts other than alfalfa do not infect alfalfa or do so poorly (Barbara DJ &Clewes E 2003;Correll JC et al.1988).This grouping is strongly supported by molecular markers(Barbara DJ &Clewes E 2003) and VCG data (Correll JC et al.1988;Gordon TR et al.1986).A number of strains morphologically described as V.alboatrum cluster separately based on the internal transcribed spacer (ITS) rDNA regions (Morton A et al.1995;Robb J et al.1993),and thus were designated as V.albo-atrum Grp2.All other strains of V.albo-atrum are referred to as Grp1 (Barbara DJ &Clewes E 2003).The resting structures and molecula rmarkers such as random amplification of polymorphic DNA (RAPDs)and ITS sequences all are distinct from these two groups (Robb J et al.1993;Mahuku GS &Platt HW 2002).Despite recognizing that the differences are significant enough to elevate Grp2 strains to a separate species,they are currently only recognized as a distinct operational taxonomic unit (Mahuku GS &Platt HW 2002).

Unlike V.dahliae and V.albo-atrum,V.tricorpus is a more powerful soil saprophyte,which may not be impeded in its germination and growth by the absence of a potential host (Isaac I 1967).Chlamy-dospores,microsclerotia,and melanized hyphae serve as survival structures for V.tricorpus.Because it is considered a weak pathogen on many hosts,research has focused on the potential for V.tricorpus to reduce the impact of Verticillium wilt induced by V.dahliae or V.albo-atrum.Co-inoculations of V.tricorpus with V.dahliae in lettuce and artichoke,and V.dahliae or V.albo-atrum in potato have resulted in a lower severity of Verticillium wilt or potato early dying symptoms,compared with plants inoculated with either of the latter two species separately (Qin QM et al.2008;Robinson N et al.2007).

3.2 Morphological Characteristics of Main Pathogens Caused Verticillium Wilt of Potato

It is reported that there are 6 pathogens caused verticillium wilt of potato of which belongs to Moniliales of Hyphomycetes.V.albo-atrum,V.dahliae,V.nonalfalfae Inderb.,V.nubilum Pethybridge,and V.tricorpus Isaac belong to Moniliaceae Verticillium.But G.nigrescens (V.nigrescens) belongs to Plectosphaerellaceae Gibellulopsis.But,V.dahliae and V.albo-atrum are the most harmful to potatoes.They are the main pathogens of Verticillium wilt of potatoes and the most important plant quarantine objects in China.They can lead to the decline in potato yield and quality even the termination of potato production.V.nonalfalfae,V.nubilum,V.tricorpus and G.nigrescens have a weak parasitic or saprophytic life in the growing or storage period of potatoes,and do relatively little harm to potatoes.(Wang.M.X.2010;EBIHARA Y et al.2003;HARVEY C S 1965;INDERBITZIN P et al.2011;ISAAC I 1949,1953)。

3.2.1 V.dahliae

The colony was initially white and flocculent on PDA medium.And then,after 1 week,a black,thick-walled,dormant sclerotoid,also known as microsclerotia,was formed on the colony,with a diameter of 30.0 -60.0(215.0)μm,but no dormant,septum dark mycelium tracts were produced.After 2-3 weeks,the whole colony turned black,and the diameter of the colony was 4.0-6.0cm at 2 weeks.Hyphae is colorless,slender,and have the diaphragm,which is similar to v.albo -atrum.The size of conidiogenous cell was (13.7-21.4) ×(2.3 -2.7) μm,and the top layer cells were enlarged and tapered at the bottom,with a diameter of 2.0 -4.0 μm and terminal 1 to several conidia per branchlet.The base of conidiophore is always transparent and dark,usually composed of 2 -4 layers of wheel and an apical branch,occasionally 8 layers,the length is 110.0 -130.0 μm,each layer is 20.0 -45.0 μm,each round is 3 -4(5) branch.Spores are colorless,unicellular,spherical,elliptic,obovate,ovate or fusiform,with size [2.3 -5.5(9.1)×(1.5 -3.0)) μm (Table1.).

3.2.2 V.albo-atrum

The colony is white,dense,and titled in PDA,but after 2-3 weeks,the mid-back of the colony is grayish black.the diameter of colony was 4.5 -5.5cm at 2 weeks.Air hyphae developed,and the nutritive bodies were septum mycelium,with the diameter of (1.0)2.0-4.0 μm,transparent or light color,thin cell wall.Dormancy hyphae can aggregate to form nodular mycelia,but does not produce microsclerotia.Conidiogenous cells are mostly 3 -4,and are straight or slightly curved,septum at base.The size is always 3.0 -4.0 μm wide,tapering 1.0 μm wide upward,24.0 -32.0 μm long.The length of sporogenic cells at the terminal was 30.0-46.0 μm.About conidiophore,radial and dark mycelia form at the base,which is its unique feature,but this feature is easy to disappear after repeated transfer of artificial medium,and conidium will become slightly longer and narrower.Spores are achromatous,oblong.The size of the first formation was (6.0 -12.0) ×(2.5 -3.0) m,and the spores became slightly smaller due to the increase of sporulation,which was (3.0 -7.0) ×(1.5 -3.0) m,occasionally with a transverse septum (Table1).

Table 1 Morphological characteristics of V.dahliae and V.albo-atrum

4 Control Measures of Verticillium Wilt of Potato

Verticillium wilt is also known to occur in conjunction with several other pests,pathogens,and diseases,including root lesion nematode,black dot (caused by Colletotrichum coccodes),Fusarium spp.,and Erwinia spp.,forming a disease complex known as potato early dying (Dervis S et al.2007;Devay JE et al.1974).Control of the Verticillium wilt pathogen is particularly difficult due to its wide host range and the production of hardy resting structures called microsclerotia that can persist in the soil for over a decade (Devay JE et al.1974;Douhan LI &Johnson DA.2001).Traditionally,control has focused on reducing the populations of microsclerotia in the soil,primarily through the use of chemical fumigants such as methyl bromide and metam sodium.However,the high cost,potential negative health and environmental effects,and somewhat inconsistent efficacy of fumigation,as well as the worldwide phaseout of several effective fumigants,has led to increased attention on alternative methods of control for this disease(Atallah ZK et al.2007;Devay JE et al.1974;Daayf F 1995).

Previous research on the use of organic amendments,including animal and green manures,and diseases uppressive crop rotations,has led to some very interesting and promising results.The use of soil amendments and non-chemical approaches to the management of Verticillium wilt have been the subject of recent reviews (Bhat RG &Subbarao KV 1999;Collado-Romero M et al.2006;Collins A et al.2005).

According to Larkin R P et al.（2011）,the ability of disease-suppressive rotation crops to reduce potato disease and increase crop productivity in a field with prior severe Verticillium wilt,as well as the potential influence of previous cropping history on disease suppression,was evaluated over three field seasons in Maine.Disease-suppressive rotations consisted of:(i) a highglucosinolat mustard blend (‘Caliente 119’) as a mixture of white mustard (Sinapis alba) and oriental mustard (Brassica juncea)with known biofumigation potential and (ii) a sorghum-sudangrass hybrid.Each was grown as single-season green manures followed by a subsequent potato crop.These rotations were compared with a standard barley rotation and a barley rotation followed by chemical fumigation with metam sodium as controls.Both green manure rotations significantly reduced (average reductions of 25 and 18%,respectively) Verticillium wilt in the subsequent potato crop compared with the standard barley control but were not as effective as chemical fumigation (35% reduction).The mustard blend also reduced other soil-borne ases (black scurf and common scab) better than all other rotations.Mustard blend and chemical fumigation treatments increased tuber yield relative to the barley control by 12 and 18%,respectively.However,by the second rotation cycle,disease levels were high in all rotations,and only chemical fumigation resulted in substantial disease reduction(35%).Rotations also had significant effects on soil micro-biology,including soil bacterial and fungal populations and microbial community characteristics based on fatty acid profiles.However,only chemical fumigation significantly reduced soil populations of Verticil-lium spp.and increased general soil microbial activity.Previous cropping history did not significantly affect disease reduction,tuber yield,or soil microbial communities.This research indicates the potential for using disease-suppressive rotations for managing Verticillium wilt and other soil-borne diseases but also indicates that multiple years of disease suppressive crops may be needed to substantially reduce disease in heavily infested fields(Larkin R P et al.2011).

According to Naraghi L et al.（2014）,three isolates of Talaromyces flavus (TF-Po-V-49,TF-Po-V-50 and TFPo-V-52) with the most inhibitory effect on Verticillium wilt disease in the greenhouse were selected for evaluation in the field.Rice bran was used for the preparation of different inoculums affected by the above-mentioned isolates.For application in the field,potato tubers were coated with tree different inoculums.During 2011 and 2012,field experiments were carried out in randomized complete block design with five treatments and four replications in the agricultural research station of Hamedan province of Iran.Treatments included:1-3:each inocolum affected by one of three isolates of T.flavus;4:Carbendazim fungicide and 5:control without inoculum and fungicide.According to the results of this study,TF-Po-V-52 was the most effective fungal antagonist in decreasing Verticillium wilt disease severity and incidence(18.04 and 40.31% respectively) and increasing yield of potato(17.48%) and could be a potential biofungicide and biofertilizer in the fields.Statistical groups of the treatments in terms of average yield showed that the yield increased from 3.69 to 26.27% in all treatments compared to the control group.Among all treatments,those affected by carbendazim and TF-Po-V-52 showed the most efficacy in increasing yield (28.74 and 26.74 ton/ha respectively)in comparison with the control group(22.76 ton/ha) (Naraghi L et al.2014).

Simko I et al.（2017）have evaluated the reaction of 283 potato clones (274 cultivars and nine breeding selections)to inoculation with V.dahliae under greenhouse conditions.A significant linear correlation (r=0.4,p ＜ 0.0001) was detected between plant maturity and partial resistance to the pathogen,with late maturing clones being generally more resistant.Maturityadjusted resistance,that takes into consideration both plant maturity and resistance,was calculated from residuals of the linear regression between the two traits.Even after adjusting for maturity,the difference in the resistance of clones was still highly significant,indicating that a substantial part of resistance cannot be explained by the effect of maturity.The highest maturity-adjusted resistance was found in the cv.Navajo,while the most susceptible clone was the cv.Pungo.They hope that the present abundance of data about the resistance and maturity of 283 clones will help potato breeders to develop cultivars with improved resistance to V.dahliae (Simko I&Haynes K G 2017).

Haiyuan,Li,et al.（2019）evaluated 30 commercially popular potato cultivars against Verticillium dahliae strainVdp83 and Verticillium nonalfalfae strain Vnp24,two well-characterized strains causing Verticillium wilt of potato in China.Both strains were isolated from diseased potato plants,and they were previously proven to be highly virulent.Ten plants of each cultivar were inoculated with the V.dahliae strain and incubated on greenhouse benches.Symptoms were rated at weekly intervals,and the relative area under the disease progress curve was calculated.The experiment was repeated once,and nonparametric analysis was used to calculate the relative marginal effects and the corresponding confidence intervals.Five resistant cultivars and four susceptible cultivars identified from the analyses were then challenged with the V.nonalfalfae strain.Cultivar responses to V.nonalfalfae were like those exhibited against V.dahliae,except for one cultivar.This study showed that resistance among potato cultivars exists in China against Verticillium spp.and that the resistance to V.dahliae identified in potato is also effective against the other Verticillium species that cause Verticillium wilt with a few exceptions.Cultivars identified as resistant to Verticillium wilt can be deployed to manage the disease until the breeding programs develop new cultivars with resistance from the sources identified in this study (Haiyuan,Li et al.2019).

5 Concluding Remarks

The potato industry is an important part of high quality,high efficiency and foreign exchange -generating ecological agriculture.In recent years,potato industry has been paid more and more attention by an increasing number of countries,especially developing countries.It has played a crucial role in ensuring national food security and promoting the development of rural economy and increasing the income of farmers.China is the largest producer of potato in the world and Inner Mongolia is the main region producing potato in China.However,in recent years,with the development of potato industry,continuous cropping years in potato planting areas have been continuously extended,and soil-borne diseases have become increasingly severe,which has become one of the main obstacles to potato production.In particular,verticillium wilt of potato has become one of the most common potato diseases in Inner Mongolia.Therefore,it should arouse the attention of researchers and related departments to study the disease comprehensively and systematically.It is of great significance for the sustainable development of potato industry to strengthen the management of potato variety selection,agricultural operation,biological control and so on,and to implement active and effective control measures for the verticillium wilt of potato.