Fenglan LI,Yao FU,Qiang YUAN,Wenhe LU,Yongqing XU,Rongmei LIU,Baozhong HU,Fuwei CHEN,Yuanyuan XU,Yanzhong FENG

1.College of Life Science,Northeast Agricultural University,Harbin 150030,China;2.Heilongjiang Academy of Agricultural Sciences,Harbin 150086,China

Potato,the main crop for food,vegetable,animal feed and industrial raw materials,has high yield,wide adaptability,high economic efficiency and abundant nutrients,and has contributed much to the food supply and safety in China’s underdeveloped areas.It also plays an important role in post-disaster relief,adjustment of agricultural structure and regional distribution[1].In recent years,potato has been planted in an increasing scale,and has become China’s forth most important food crop.China now is the largest potato planting country all over the world,producing 1/4 of world’s potato[2].In Northern China,both commercial and seed potatoes are mainly stored in cellars in winters,while the inappropriate storage conditions often result in some disease to potatoes,such as late blight,ring rot,dry rot,black heartand softrot,severely affecting potato yield and quality,as well as potato production and sales in the next year[5].Various diseases of potato emerge with the planting area increasing,and now have become the main factor limiting the development of potato industry.Among them,Fusariumdry rot is one of the main potato diseases at storage period,and causes 60% loss of potato stored in cellars[6-7].

Most previous studies about potato dry rot focused on the isolation and identification of pathogens and disease prevention,but the interaction mechanism between pathogens and potato is still unclear[8].The genetic characteristics,variations in physiological and biochemical properties of potato tubers duringF.trichothecioidesinfection have been rarely re-ported so far[9-11].At present,there are only some simple chemical methods forthe prevention and controlofFusariumdry rot in potato,and all the methods can only reduce the damages.However,they can not avoid the incidence of the disease,but also causes serious environmental pollution.So far,researchers have not found an effective method for the prevention and control of potato dry rot,because of the lack of theoretical basis.The interaction mechanism between potato andF.trichothecioidesis the basis to select an effective way to prevent and control potato dry rot[12-14].

Potato dry rot is caused by various pathogens of genusFusarium,and those pathogens can grow in a wide range of temperature 5-30℃and humidity.High temperature and humidity are most conducive to dry rot incidence,particularly under poor storage and ventilation conditions[15].It is reported that the occurrence ofFusariumdry rot is more frequent in earlymaturing potato varieties than in latematuring varieties[16].According to statistics,there are six species or variants causing potato dry rot in Heilongjiang Province,they areFusariumsambucinum,F.avenaceum,F.trichothecioides,F.solanivar.coeruleum,Fusarium solani,F.sporatrioidesandF.culmorum,respectively.Among them,F.sambucinum,F.avenaceumandF.trichothecioidesare more pathogenic[17-18].In this study,the activities of main antioxidases and the content of malondialdehyde(MDA)in potato tubers infected withF.trichothecioideswere measured,to determine the roles of the defense enzymes duringF.trichothecioidesinfection,and to provide theoretical basis for the scientific control ofFusariumdiseases,and the breeding of new potato varieties with broad-spectrum disease resistance.

Materials and Methods

Materials

The detoxified tubers of two major potato varieties in Heilongjiang Province Keshan 1(resistant toFusarium trichothecioides),Atlantic(susceptible toF.trichothecioides)were used as the host materials.F.trichothecioidesWollenw was preserved in PDA medium in our laboratory.

Methods

The solid culture medium ofF.trichothecioidesWollenw was punched with a hole punch to obtain 1 cm disks.Potato tubers of Atlantic and Keshan 1 were surface sterilized,peeled,and sliced into pieces 5 mm in thickness and 3 cm in diameter.Then,pathogen disks were inoculated into the center of each potato pieces in dishes,and cultured at 25℃.1.0 g of the samples were collected 3,6,12,24,48 and 72 h later,wrapped with foil and immediately placed in liquid nitrogen.Three repetitions were set for each sampling.After that,all the samples were measured through enzymeprotein assay for target physiological and biochemical indices[19].

Results and Analysis

Variations in soluble protein content during F.trichothecioides infection

The tubers of Atlantic and Keshan 1 were infected byF.trichothecioides.During this process,the soluble protein content in the tubers was measured at different time.As shown in Fig.1,the soluble protein content in Atlantic changed over time afterFusariuminfection,peaked and exceeded the uninfected control at hour 9,and was below the control at other time periods.The soluble protein content in Keshan 1 tubers infected byF.trichothecioideschanged slightly compared with the uninfected control,and it was 43% higher than that of the uninfected at only hour 9.It could be concluded from Fig.1 that the soluble protein content in the infected tubers of the two potato varieties changed in a similar pattern,lower than that of the uninfected at most time.In addition,the soluble protein content of Atlantic which is susceptible toF.trichothecioides,was higher than that of Keshan 1,which is resistant toF.trichothecioides.The results suggested that the soluble protein content may be negatively correlated to the resistance of potato varieties toF.trichothecioides.

Variations of SOD activity during F.trichothecioides infection

As shown in Fig.2,the SOD activity of both potato varieties decreased 3 h after infection and then rose sharply.The SOD activity in infected Keshan 1 peaked at hour 6,that in infected Atlantic peaked at hour 9.From hour 9 to 48,the SOD activity in infected Keshan 1 which is resistant toF.trichothecioidesincreased again,while that in infected Atlantic which is susceptible toF.trichothecioideskept decreasing.The results indicated that the increasing SOD activity in infected potato tubers had a certain relationship to the resistance of potato varieties toF.trichothecioides.

Variation of POD activity during F.trichothecioides infection

As shown in Fig.3,the POD activity in infected Atlantic and Keshan 1 tubers decreased within the first 6 h,while that in Atlantic changed more significantly.6 h after infection,the POD activity in both potato varieties began to increase.In addition,the POD activity in Keshan 1 which is resistant toF.trichothecioideswas always higher than that in Atlantic which is susceptible toF.trichothecioideswithin the 12 h after infection.The results proved that the high POD activity is closely related to the strong resistance of potato varieties toF.trichothecioides.

Variation of malondialdehyde(MDA)content during F.trichothecioides infection

As shown in Fig.4,compared with the control uninfected,the MDA content in infected Keshan 1 tubers decreased slightly from the beginning,and greatly at hour 24;the MDA content in infected Atlantic tubers decreased significantly after infection,and more significantly at hour 12.A large number of antioxidants were generated after fungal infection,which might be the reason causing the decrease in MDA content.

Discussion

Reactive oxygen species(ROS)is a very important part in plant defense response,as it can trigger programmed cell death(PCD),drive the oxidative crosslinking of cell wall proteins,so that the pathogens can be confined to the suicidal host cells,thereby slowing down pathogen invasion[20].A free radical reaction may be triggered in the plants under stress.The study by Huet al.[21]revealed that the SOD activity inAcer truncatumunder simulated drought stress decreased at first and then gradually increased to normallevel.Previous studies have shown that the SOD activity in the plants resistant to drought,frost,high salinity and toxics is significantly higher,indicating that high SOD activity is related to plant’s resistance to stresses,facilitating the selection of new varieties with strong resistance and high economic benefits[22].Peroxidase (POD),a ubiquitous and active enzyme in plants,is closely related to plant metabolism and resistance[23-24].It has a dual role:it has a protective role at early period of aging and response to stress,and then it is involved in the generation of reactive oxygen species at late period of aging and response to stress.It is a product of plant aging,and even can be used as an index of senescence[25].Malondialdehyde(MDA)is a most commonly used index for the measurement of membrane lipid peroxidation[26].The damages caused by stress or aging to plants are closely related to reactive oxygen-induced membrane lipid peroxidation.Diene conjugates,lipid peroxides,MDA and ethylene,etc.are the products of membrane lipid peroxidation,among which,MDA is one of the most important one.So MDA content can be used as an index for measuring the degree of membrane lipid peroxidation,thereby indirectly determining the damages in membrane system and plant resistance to stresses.

In this experiment,we found that the soluble protein content inF.trichothecioidesinfected Atlantic and Keshan 1 tubers were lower than that of the uninfected;the soluble protein content of infected Atlantic which is susceptible toF.trichothecioideswas higher than that of Keshan 1,which is resistant toF.trichothecioides,indicating that soluble protein content is negatively correlated to the resistance of potato varieties toF.trichothecioides.From 9 to 48 h afterF.trichothecioidesinfection,the SOD activity of Keshan 1 began to increase,while that of Atlantic kept decreasing,suggesting that SOD activity has a certain relationship with the resistance of potato varieties toF.trichothecioides.The POD activity in both potato varieties changed significantly afterF.trichothecioidesinfection,proving that it is closely related to the resistance of potato varieties toF.trichothecioides.We also found that the MDA content in potato tubers also significantly reduced a certain time period afterF.trichothecioidesinfection,which indicated that MDA content may have a certain role in disease resistance of potato.

The defense enzymes play a crucial role in potato’s resistance toFusariumdry rot.The activities of related defense enzymes inF.trichothecioides-resistant potato varieties increased at varying degrees afterF.trichothecioidesinfection,indicating that these enzymes are related to potato’s resistance toF.trichothecioides.This paper illustrates the physiological basis of potato’s resistance according to the changes in antioxidase activity and provides a theoretical basis for the selection of disease-resistant potato varieties.

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