The Control Research Progress ofLiberobacter asiaticumin Taizhou City

Jihua YU,Minrong ZHANG,Jian TAO,Lu LU,Xiao YANG,Enguo WANG,Zhiyong YE

1.Plant Quarantine Stations of Huangyan District,Huangyan 318020,China;

2.Plant Protection Station of Linhai City,Linhai 317000,China;

3.Huangyan Agriculture Bureau,Huangyan 318020,China

The Control Research Progress ofLiberobacter asiaticumin Taizhou City

Jihua YU1*,Minrong ZHANG1,Jian TAO1,Lu LU1,Xiao YANG1,Enguo WANG2,Zhiyong YE3

1.Plant Quarantine Stations of Huangyan District,Huangyan 318020,China;

2.Plant Protection Station of Linhai City,Linhai 317000,China;

3.Huangyan Agriculture Bureau,Huangyan 318020,China

After decades of researches and discussions on occurrence regularity and control technology of citrus psyllid(Diaphorina citri Kuwayana)and Liberobacter asiaticum,the occurrence regularity and sampling technique of citrus psyllid in orange forests in Taizhou was defined and the connections between the infection rate and carrying rate of citrus psyllid and Liberobacter asiaticum as well as the correlations between the Liberobacter asiaticum and citrus yield loss were discussed.This paper discussed the warnings of Liberobacter asiaticum and citrus psylid control index,the economic life span of orange forests was then predicted by building the diseases spreading models with different management styles.At last,the paper put forward the comprehensive prevention and control technology and the concept that Liberobacter asiaticum was preventable and controllable.

Liberobacter asiaticum;Citrus psyllid;Occurrence regularity;Disease Spreading models;Control index;Prevention and control technology

T aizhou is located along the southeast coast of Zhejiang P rovince,and the citrus is one of the leading industries of agriculture. Huangyan mandarin orange dated from the Three Kingdoms and flourished in the Tang and Song dynasties, which had 1 700 years of cultural history before present and was the ancestor of the world’s citrus[1-4].Liberobacter asiaticum was the quarantine pest that had fastest spreading speed, heaviest damage,largest hazard and hardest prevention and treatment, meanwhile it was the international scientific and technological problems that had not yet to be conquered in the cur rent[2].Since 2002,Liberobacter asiaticum was firstly introduced to Taizhou,the citrus industry was severely threatened by this disease.A total of 1.9945 million sick trees were cut in Huangyan for 12 years[3].By a series of researches on the disease spreading,vector insect,control index, comprehensive prevention and control technology,a series of comprehensive prevention and control technology formed based on the main prevention and control of Diaphorina citri,which effectively contained the occurrence and damage of Liberobacter asiaticum and promoted the healthy and sustainable development of the citrus industry.To this end,this study revealed the relationships of Liberobacter asiaticum and citrus psyllid,the occurrence and spatial distribution of citrus psyllid,the relationships of Liberobacter asiaticum and yield loss,epidemic regularity and diffusion dynamic model of Liberobacter asiaticum and the prevention and control measures of Liberobacter asiaticum.

The Relationships ofLiberobacter asiaticumand Yield Loss

By surveying the susceptibility ofLiberobacter asiaticumin different varieties from 2003 to 2005,4 main citrus varieties in 25 citrus orchards were investigated.The results showed that the average infection rate ofCitrus reticulataBlanco cv.Tardiferax,Citrus reticulataBlanco cv.Succosa,satsuma orange and Ponkan were 18.99%,25.63%,31.09%and 39.55% respectively,disease index was 9.03,10.32,14.77 and 23.54 respectively.It means that the susceptibility ofLiberobacter asiaticumof main citrus varieties was Ponkan＞satsuma orange＞Citrus reticulataBlanco cv.Succosa＞Citrus reticulataBlanco cv.Tardiferax[5].

By surveying the relationships ofLiberobacter asiaticumand yield loss from 2002 to 2009,the healthy fruit numbers were calculated and the healthy fruit rate were compared in different illness state.The results showed that the healthy fruit rate and fruit amount of single plant decreased with the increase of disease severity[6]. The correlations between different disease severity ofLiberobacter asiaticumand single fruit weight and strains yield was negative,but the correlations with yield loss was positive,and the degree of correlation reached a significant level.The disease severity ofLiberobacter asiaticumwas higher,the single plant weight and strains yield was lower, and the yield loss was larger[7].

The Relationships ofLiberobacter asiaticumand Citrus Psyllid

Studied the relationships of the infection rate and carrying rate of adult citrus psyllid by PCR detection[8-10],the established mathematical model indicated that the correlations between the morbidity ofLiberobacter asiaticumand the carrying rate of citrus psyllid reached extremely significant level. The correlations between the amount of larvae per plants of citrus psyllid and larvae plant rate was significant.The psyllid of citrus became nymph in fall tip period was surveyed in 2006.The dates of the amount of larvae per plants of citrus psyllid and larvae plant rate of 11 orchards were analyzed, and the results showed that the correlations between the amount of larvae per plants of germ-carrying citrus psyllid and larvae plant rate was extremely significant.Meanwhile,the corresponding mathematical model was established[11].

The relationships of citrus psyllid larvae plant rate and the infection rate ofLiberobacter asiaticum.According to the occurrence condition of citrus psyllid andLiberobacter asiaticum,the results showed that in the early stage ofLiberobacter asiaticum,the disease was sporadic or light in the orchard, and the average larvae plant rate of citrus psyllid was less than 10%,which was over 10%in the orchards with middle and heavy morbidity.The correlations between larvae plant rate of citrus psyllid and the morbidity ofLiberobacter asiaticumwere extremely significant by statistical test.The orchards with higher larvae plant rate had higher morbidity,and the related equations was established in order to warnLiberobacter asiaticummorbidity by larvae plant rate[12].

The Occurrence Regularity and Distribution Characteristics of Citrus Psyllid

According to the surveys from 2003 to 2005,the occurrence generation of citrus psyllid was 6 to 7 generation in Huangyan[13].The 1st to 3rdgeneration was more orderly,and the 4thto 7thgeneration had a phenomenon of generation overlapping.The occurrence time of the 1stgeneration was in the early and middle of April,and the wintering adults began to spawn,then eclosion in the later April and early May.The eclosion time of the 2ndgeneration adult was in early July,the 3rdgeneration adult was in early August, the 4thgeneration adult was in early September and the 5thgeneration adult was in middle and later of September. The 4thand 5thgeneration occurred generation overlapping[14].The 6thand 7thgeneration were currently only found in growing stronger saplings or adult trees that the large branches full cut and the flourished late fall branch. Under normal conditions,there was no nymph after early November,but still a few adults.Currently only found adults overwinter,but not clear whether the egg or nymph live overwinter[15].

In September 2006,11 orange fields with different population density of citrus psyllid were investigated and each sample plot was surveyed and records the number of adults per plant. The results determined by aggregation degree index method[16]showed that the distribution characteristics of adult citrus psyllid were different under different population density[15].Low population density appeared low aggregation degree and high population density appeared high aggregation degree. Thus,the spatial distribution of adult citrus psyllid in the citrus was that the aggregation degree increased with the increase of population density,and accord with the negative binomial distribution[17].

Based on the analysis of citrus psyllid population distribution pattern, the optimal sampling method of the number of citrus psyllid surveying was saltatory method and five-point method[18].Meanwhile,the theoretical sampling model of adult citrus psyllid was established[19]in order to determine the sampling number table in different population density and guide prevention and control work according to the psyllid density.The determined prevention policy recommendations was:in the low-density orchards,each orchard checked 50 to 100 plants;in the medium and high density orchards, each orchard checked 20 to 50 plants, which was able to know the occurrence status of the investigated orchards[20].In order to save the investigation time,sequential sampling method could be chosen by using sequential sampling table[21].As prevention decisions of orchards,the survey should be contrast with sequential sampling table,and then stopped the investigation when the cumulative amount of insects reached predetermined indexes.Thereby calculated the average population density,whichprovided a guiding significance for early warning and prevention and control of citrus psyllid[22].

Liberobacter asiaticum vector Control Index and Early Warning Index

According to the study of the relationships between infection rate,yield loss and effective amount of citrus psyllids[23-24],the control index for psyllid in shoot stage of citrus was that the initial stage of new shoots of citrus was the optimal prevention stage for controlling and preventing germ-carrying citrus psyllid.The control index of the amount of germ-carrying citrus psyllid identified as 7.5 per one hundred plants.It means that the control index ofLiberobacter asiaticumwas 10 per one hundred plants of the amount of germ-carrying citrus psyllid.

According to the study of the relationships between infection rate and yield loss[25],the strategic control index forLiberobacter asiaticumwas established[26].Under the allowed loss level, the strategic control index was 1.0%of the infection rate of the whole orchards in the initial stage of illness.It means that if the infection rate exceeded 1.0% in the initial stage of illness,the diseased trees should be eradicated to reduce bacteria source,while overall using drug to prevent the bacteria spreading and infecting adjacent healthy trees.

According to the study of the relationships between germ-carrying rate of citrus psyllid and infection rate ofLiberobacter asiaticum,the prediction technique and early-warning index for germ-carrying rate of citrus psyllid were established.When the infection rate ofLiberobacter asiaticumin the initial stage of illness rose to 1.0%,the germ-carrying rate of citrus psyllid would exceeded 4.0%,which indicated thatLiberobacter asiaticumwould formed a large heavy diffusion damage.Thus,the germ-carrying rate of citrus psyllid could be used to make pandemic alert ofLiberobacter asiaticum.When the detected germ-carrying rate of citrus psyllid exceeded 4.0%,it needed to increase prevention and cure of citrus psyllids in the whole orchards[27-28].

Epidemic Regularity and Diffusion Dynamic Model of Liberobacter asiaticum

The distribution pattern and sampling model of Liberobacter asiaticum

Spatial distribution of Liberobacter asiaticum in premature satsuma orange variety‘Gongchuan’(hereinafter referred to as premature citrus)From 2004 to 2009,data obtained by multistage sampling survey and then determined by the aggregation degree index,the results showed that the spatial distribution pattern ofLiberobacter asiaticumin orange forests was aggregated distribution. The spatial distribution pattern changed with the aggregation intensity and disease grade.The uniformly spatial distribution pattern indicated that the disease grade in the early stage of illness was at a low level,then raised in the aggregated distribution pattern.By analyzing,the fundamental factors of aggregated distribution ofLiberobacter asiaticumin orange forests was individual group,and the orange trees attracted each other, which mainly infected and spread by germ-carrying citrus psyllid.On the basis of inference,the aggregation intensity ofLiberobacter asiaticumstrengthened with the increase of the years of incidence orchards,and increased with the rising of disease grade[29-31].

Spatial distribution of Liberobacter asiaticum and disease spreadAnalyzed by aggregation average test, the threshold that discriminated the cause ofLiberobacter asiaticumaggregation was 2.118 4 of the average disease grade density.The relationships between average aggregation intensity and average disease grade density ofLiberobacter asiaticumwere linear correlation.When the disease grade at low density,aggregation may be caused by infection of germ-carrying scion or germ-carrying psyllid or unconspicuous diseased trees.While at high density,aggregation may be caused by the spread of infection of germ-carryingLiberobacter asiaticum,which resulted in the opportunity of re-infection increased,and the average aggregation value also became larger[32-35].

Using the mathematical model via theoretical sampling to guide disease control and monitoringEpidemic prevention and control surveyed by analyzing and establishing theoretical sampling model and determining the theoretical sampling tables[36].Contrast to the theoretical sampling tables, the epidemic general investigation rate should not less than 80%.For epidemic surveillance and investigation, referred to theoretical sampling tables, 10 plants per land were checked in high density orchards;15 plants per land were checked in medium density orchards;20 to 30 plants per land were checked in low density orchards. So the morbidity status of investigated orangery could be knew[37-38].The sequential sampling table should be used to determine the survey[39-41]as to give the prevention decision of orchards.When the accumulative disease grade of the surveyed orchards reached the set index,the investigation could be stopped.The accumulative disease grade divided the total number of samples was the average disease grade density,which had an important role in prevention and monitoring and early warning ofLiberobacter asiaticum.

Epidemic diffusion regularity and dynamic model of Liberobacter asiaticum

From 2002 to 2011,orchards controlled by three different control measures contained lose control Orangerie,general prevention Orangerie,comprehensive prevention and control Orangerie were investigated in order to study the epidemic diffusion model and the prevention and control effects[42-43]in Huangyan district.The epidemic diffusion model ofLiberobacter asiaticumin lose control Orangerie showed a linear increasing trend,the diffusion rate was fastest after the invasion of 3 to 7 years,and the annual infection rate increased 9.03%-29.85%.By statistical analysis, the relationships between annual sequence and epidemic diffusion index (disease infection rate)was extremely significant.According to this,the relationship mathematical mode established.Predicting from this mode,the epidemic diffusion rate ofLiberobacterasiaticumin the loss control Orangerie from invasion to 100%of infection rate just needed nine years,that is the annual morbidity of Liberobacter asiaticum in loss control Orangerie which was not in prevention and control status was 11.11%.The epidemic diffusion model ofLiberobacter asiaticumin general prevention Orangerie showed a rapid diffusion trend,the diffusion rate was fastest after the invasion of 3 to 6 years,and the annual infection rate increased more than 7%. By research and analysis,the relationships between the annual sequence and accumulative infection rate of theLiberobacter asiaticumwere extremely significant.From this established mathematical mode,the epidemic diffusion rate ofLiberobacter asiaticumin the general prevention Orangerie from invasion to 100%of infection rate needed no less than 18 years.The actual survey indicated that the annual infection rate was 4.69%in nearly 11 years.Compared with the infection rate ofLiberobacter asiaticumin loss control Orangerie,the average control effect maintained at 40%to 50%,and the average control effect was 43.93%.According to the data surveyed in comprehensive prevention and control area,the annual sequence and accumulative infection rate were used to test its correlations,and established mathematical mode under comprehensive prevention and control conditions.The model showed that the annual morbidity ofLiberobacter asiaticumin comprehensive prevention Orangery was 0.31%.Compared with the loss control Orangerie,the average control effect ofLiberobacter asiaticumwas 96.15%[44].Therefore,in the future prevention and control work ofLiberobacter asiaticum,theLiberobacter asiaticumcould be controlled as long as make great efforts to implement the comprehensive measures.

Comprehensive Prevention Study ofLiberobacter asiaticum

Pest and disease prevention and control effect

From 2003 to 2009,the occurrence conditions of the pest control area and the loss control Orangerie were investigated.In 2009,the accumulative infection rate ofLiberobacter asiaticumwas 58%in the pest control area,which was 99.86%in the loss control Orangerie.The control effect was 41.92%in pest control area[44].

Fitness cultivation and the reduce of incidence effect

From 2003 to 2006,the occurrence conditions ofLiberobacter asiaticumin fitness cultivation orchards and general cultivation orchards were investigated.The growth of fitness cultivation orchards was obviously stronger than the control area.And the disease incidence was significantly inferior to the control area.410 orange trees in fitness cultivation orchards were investigated,18 trees were diseased,and the morbidity was 4.3%. While 270 orange trees in the control area of the adjacent orchards were investigated,44 trees were diseased, and the morbidity jumped to 16.3%[45].Comprehensive control practices ofLiberobacter asiaticumand its effects

Since November 2002,Liberobacter asiaticumwas first discovered, the government of Huangyan district paid high attention to the epidemic prevention and control work.According to the prevention and control strategies of"pay equal attention to dig and cure and manage,comprehensive prevention and control"[46-47],"6 persist"and"5 not loss"should be done to effectively controlledLiberobacter asiaticum.The incidence had been reduced to 43 thousand in 2013 from 356 thousand in 2005.A total of 1.994 5 million diseased trees were eradicated in 11 years.The occurrence range shrunk year by year,and the incidence also decreased year by year.It means that the prevention and control work obtained actual effect[48].

Problems and Prospects

Liberobacter asiaticumwas quarantine pest in China,the citrus was a kind of perennial fruit,and its economic life span was up to 100 years.The yield of 30 years-old orange trees was up to 150-200 kg per plant.But once infected byLiberobacter asiaticum, even if cut off the infected branches, the life span of the orange trees was less than 5 years,and the life span of orchards was less than 10 years[49]. The pathogenetic fruits had no edible value,so farmers suffered heavy losses.Today’s technology could not solve the problem ofLiberobacter asiaticum,the measures were only cut and dig trees.From 10 years of practice for the occurrence regularity research and prevention and control work ofLiberobacter asiaticum,as long as government leadership and orange farmers did teamwork actively, remould the heavy pathogenetic orchards,planted virus-free seedlings, adopted comprehensive prevention and control measures,grasped the prevention and control work in the critical period,eradicated the primary infection trees,Liberobacter asiaticumwas preventable and controllable.Of course,with the help of modern molecular biology techniques to study the interaction mechanism of citrus themselves andLiberobacter asiaticum,searched the anti-diseaseresistant gene[50],cultivated and screened resistant varieties,or transferred resistance gene to an existing highquality varieties without changing the quality,which made the citrus do not morbidity or show symptoms,maybe could fundamentally solve the problem of prevention and control ofLiberobacter asiaticum.

In addition,if made a breakthrough at isolated culture of pathogens and treatment technology, not adopted cut and dig measures to solve pathogenetic or infectious citrus, but took chemotherapy treatment to make citrus recover healthy gradually. In this way,cut and dig rate of diseased trees would reduce greatly and the costs of prevention and control work would significantly reduced.For this reason,the recent researches of treatment technology ofLiberobacter asiaticumattempted to adopt biologic agents such as deguelin,flavonoids and terpenoids.The achieved initial results indicated that the action principle of such agents had relatively strong absorption and penetration,which could penetrate to the interior of vascular tissue of citrus and killedLiberobacter asiaticum.Meanwhile,it could softened the impurities in the screen wall,dredged guide screen pipe,andensured normal transport of water,nutrient and leaf photosynthesis products.In the top of graft union of citrus stem base,with a diameter of 3.5-4.0 mm drill bit and angled 30°-35°,cross drilling 3 to 4 holes with the depth of 2-4 cm according to the size of citrus to make the drug liquid evenly inject into the tree body.And then used a kind of fruiter drug liquid injection device[51]. The prepared bactericide and dredge agents were injected into diseased trees in vivo with high-pressure method,which not only killed infected pathogens in the trees body,but also dredged transport corridors blocked by pathogens,and made nutrients and water of citrus transport unblocked, restored growth function of sick citrus, reached therapeutic purposes ofLiberobacter asiaticum.By means of modern molecular biology to find resistance genes,cultivating and screening resistant varieties and taking medication technology will help to finally solveLiberobacter asiaticum, which perhaps the ultimate goal that scientists do the study ofLiberobacter asiaticum.

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Responsible editor:Lin FANG

Responsible proofreader:Xiaoyan WU

臺(tái)州柑橘黃龍病防控研究新進(jìn)展

余繼華1，張敏榮1*，陶健1，盧璐1，楊曉1，汪恩國2，葉志勇3
（1.浙江省臺(tái)州市黃巖區(qū)植物檢疫站，浙江黃巖318020；2.浙江省臨海市植物保護(hù)站，浙江臨海317000；3.浙江省臺(tái)州市黃巖區(qū)農(nóng)業(yè)局，浙江黃巖318020）

根據(jù)十幾年對柑橘木虱和柑橘黃龍病發(fā)生規(guī)律與防控技術(shù)研究探討，明確了柑橘木虱在臺(tái)州橘林的發(fā)生分布規(guī)律和抽樣技術(shù)，分析了柑橘木虱蟲株率和帶毒率與黃龍病、黃龍病與柑橘產(chǎn)量損失等之間的關(guān)系，探討了柑橘黃龍病預(yù)警和柑橘木虱防治指標(biāo)，建立了不同管理方式下的疫情擴(kuò)散模型預(yù)測橘林經(jīng)濟(jì)壽命，提出了柑橘黃龍病綜合防控技術(shù)和可防可控的理念。

柑橘黃龍病；柑橘木虱；發(fā)生規(guī)律；擴(kuò)散模型；控制指標(biāo)；防控技術(shù)

農(nóng)業(yè)部公益性行業(yè)（農(nóng)業(yè)）科研專項(xiàng)經(jīng)費(fèi)項(xiàng)目“柑橘黃龍病和潰瘍病綜合防控技術(shù)研究與示范”（201003067）；浙江省科技計(jì)劃項(xiàng)目“柑橘黃龍病監(jiān)測與防控策略研究”（2004C32087）。

余繼華（1960-），男，浙江臺(tái)州人，高級(jí)農(nóng)藝師，碩士，從事植物檢疫與防控技術(shù)研究。*通訊作者，E-mail：yo5428@zjip.com。

2015-03-08

修回日期 2015-05-10

Supported by the Ministry of Agriculture and public service sectors(agriculture) Research and Special Project Funds‘Comprehensive Prevention and Control Technology Research and Demonstration of Liberibacter americanu s and Canker’(201003067);Science and Technology Planning Project of Zhejiang Province‘Research of Surveillance and Control Strategy of Liberibacter americanus’(2004C32087).

*Corresponding author.E-mail:yo5428@zjip.com

Received:March 8,2015 Accepted:May 10,2015