【作者】 汪志辉；

【导师】 廖明安；

【作者基本信息】 四川农业大学 ， 果树学， 2008， 博士

【摘要】 柑桔是世界范围内的重要果树,全世界有80多个国家栽培柑桔,其栽培面积和产量均居世界首位。我国是柑桔主产国,主要分布在长江流域及以南地区,经济栽培的省有19个,集中在北纬20°～30°、海拔700-1000m的缓坡及丘陵地带。栽培面积近年稳定在120万～130万hm²,产量居世界第三位。从目前柑桔产业发展来看“易剥皮、无核（少核）、有香味、风味浓”已成为发展趋势,杂交柑（主要是桔与橙、桔与柚的杂交后代）正是迎合了这一趋势,近年来发展迅速。‘不知火’（C.unshiu×C.sinensis cv.Skiranui tangerine）是近年中国农科院柑桔研究所从日本引进推广的杂交柑良种之一,是21世纪杂交柑主推品种。该品种的特点是:果大无核,平均单重250g左右;可溶性固形物高达16-18%,果肉细嫩脆,且特别化渣;汁多酥甜,风味浓,品质极优。成熟期在每年的2～3月,正值水果淡季;因其优良的品质、早果、丰产及晚熟等特性,在我国南方特别是四川盆地得以迅速推广发展。四川目前杂交柑栽培面积已达6667hm²,其中‘不知火’所占比例达到60%以上。我国南方地区,特别是四川盆地近年来由于气候的变化,每年冬季及翌年早春的低温尤其是霜冻害越来越频繁;而‘不知火’果实的成熟时间为2～3月,当果实在接近成熟的时候往往遭受四川盆地最冷月（12月～翌年1月）的霜冻威胁,造成大量落果,产量锐减。在生产上普遍采用的方法是在最冷月来临前树冠覆盖薄膜,此法虽可在一定程度上减轻果实霜冻害,但由于在果实糖分转化的关键时期对树冠严重遮光,此时,又正值四川盆地全年光照最弱的时期,从而导致树冠光照条件严重恶化,果实品质严重下降。目前,关于柑桔霜冻害研究的文献报道均着重于叶和枝干方面,而有关柑桔果实霜冻害的研究,特别是关于由INA细菌（Ice Nucleation Active Bacteria,简称INA细菌）引起‘不知火’果实的霜冻害及其防治方法的研究,均未见报道。本研究以‘不知火’杂交柑果实为试材,通过分离、纯化其果实上可能存在的优势INA细菌,并加以鉴定;利用化学试剂和从同生态条件下抗霜冻能力较强的‘伏令夏橙’（C.sinensis cv.Valencia orange）、红玉血橙、枳、酸柚等果实上分离出的拮抗菌,对‘不知火’果实上的INA细菌进行室内和田间防除试验;以期寻找一条既不降低‘不知火’杂柑果实品质,又能安全有效地防治霜冻危害的方法,为在该气候条件下生产出高品质的‘不知火’杂柑提供可靠保障。研究的主要结果如下:（1）采用平板稀释分离技术和Vail小液滴冻结法,首次在‘不知火’果实表面分离得到9株INA细菌;以中国农科院植物保护研究所植物病虫害生物学国家重点实验室分子植病课题组提供的凤梨欧文氏菌INA110（Erwinia ananas）和丁香假单胞菌INA3023（Pseudomonas syringae pv.syringae）作为标准菌株,通过经典的伯杰氏细菌学鉴定程序,确定4株为凤梨欧文氏菌（E.ananas）,占44.44%;5株为丁香假单胞菌（P.syringae pvs）,占55.56%。（2）从‘不知火’果实上分离、纯化的INA细菌,其冰核活性受温度、菌液浓度及pH值的影响。若INA细菌密度为5×10²～5×10⁸个·ml^-1,温度在-2～-7℃范围内,菌液密度越高冰核活性越强;当菌液密度一定时,温度越低冰核活性越强。INA细菌生长的pH值范围为5.0～9.0,最适为7.0,pH值为2～4的酸性溶液或pH值为10的碱性溶液都会破坏INA细菌的冰核活性。供试的抗霜素1号、石硫合剂、代森锰锌三种化学药剂对菌悬液中的INA细菌防除效果均在90%以上:其中又以抗霜素1号（2000倍）防除效果最好,其对INA细菌菌株的杀灭率在6h便达到99.80%以上。（3）在模拟自然霜冻的人工霜箱中,抗霜素1号（1000倍液、1500倍液、2000倍液）、80%代森锰锌（500倍液）、72%农用链霉素（1000倍液）、99.5%分析纯硼酸（200倍液）、98%分析纯铜铁试剂（1000倍液）、98%分析纯锌试剂（1000倍液）,以及用石硫合剂和硼酸调制pH值为10和4的十种不同的化学药剂处理中,以抗霜素1号（2000倍）对‘不知火’果实活体上的INA细菌杀灭效果最好;有效杀灭率为99.80%。（4）在同一生态条件下的‘伏令夏橙’（C.sinensis cv.Valencia orange）、红桔、枳、酸柚、红玉血橙五种果实上,分离出16株对INA细菌有拮抗作用的拮抗菌;其中,以‘伏令夏橙’上分离的D₁菌株在实验室条件下对INA细菌的拮抗性最强。（5）在温度低于0℃的时间为14d,有霜天为8d,有雪天为5d,绝对最低温度在-5℃的条件下;抗霜素1号（1000倍液、1500倍液、2000倍液）、80%代森锰锌（500倍液）、72%农用链霉素（1000倍液）、99.5%分析纯硼酸（200倍液）、98%分析纯铜铁试剂（1000倍液）、98%分析纯锌试剂（1000倍液）,以及用石硫合剂和硼酸调制Ph值为10和4的十种药剂田间处理中,以抗霜素1号（2000倍液）和硼酸（200倍液）的防霜效果较好,两种处理的冻伤率分别为8.90%和10.00%。从‘伏令夏橙’、红桔、枳、酸柚、红玉血橙五种果实上筛选的六种拮抗菌(D₁、D₂、D₅、D₇、D₉、D₁₂)对‘不知火’果实的田间防霜效果均不理想。但仍以‘伏令夏橙’果实上分离的D₁、D₂的拮抗效果相对较好。在塑料薄膜大棚、塑料薄膜小拱棚、塑料薄膜树冠浮面覆盖、果实套袋（白色）、果实套袋（内黑外灰）、果实套袋（牛皮纸）六种不同的物理防霜处理方法中,以塑料薄膜大棚处理的田间防治效果最好,该处理的‘不知火’果实冻伤率为5.57%;但塑料薄膜大棚造价高,在生产中难以推广。在上述药剂、生防菌、果实套袋以及设施处理等22种不同的田间防霜处理中,以设施处理对‘不知火’树体的花芽分化和果实品质影响较大,塑料薄膜树冠浮面覆盖处理的有叶单花率仅为5.03%;结合对果实品质的影响,成本控制,操作的难易程度以及对第二年产量影响等综合因素考虑来看,以抗霜素1号（2000倍液）、硼酸（200倍液）和果实套袋（白色）处理的防霜效果理想,可以在生产上推广应用。更多还原

【Abstract】 Citrus was one of the important fruit trees.Citrus were cultivated by more than 80 countries in the world.And the area and output of citrus was premier in the world.China was the primary to plant the citrus,mainly distributed in the Changjiang River and the southern area.There were 19 provinces to plant it,and concentrated in the gentle slope and the foothill which was the north latitude 20°～30°and the elevation 700～1000m.In Recent years,the cultivated areas were stabilized in 120～130 hm² ten thousand,and the outputs were the third in the world.From currently the citrus industry development,it had become the development trend which was" peel easily,aspermous（little pit）,had the thick flavor,taste".The hybrid citrus（orange and orange,orange and pomelo） would cater to this development trend,and could develop quickly in recent years.The Skiranui tangerine（C.unshiu×C sinensis cv.Skiranui tangerine） was one of the superior hybrid citrus cultivars that were introduced and extended by the Citrus Institute in Institute of Agricultural Science in china from Japan.It was seen as citrus cultivars popularly in the 21 st century.The cultivars had more characteristics that the fruit was big and aspermous,average each weight was about 250 grams,dissoluble solid content was up to 16～18%,flesh was tender and crisp,the taste was thick,and the quality was very excellent.And its mature period was the annual February-March that was off-season in the fresh fruit market.In sichuan,hybrid citrus cultivars developed quickly because of its quality,output and so on.The cultivated area of hybrid citrus cultivars was 6667hm² at present,and the Skiranui tangerine was above 60%.In the south of our country,especially Sichuan Basin,in recent years because of the climatic change,the low temperature of the annual winter and the early spring next year were more and more frequent.However the mature time of the Skiranui tangerine was February to March.When nearly ripe,the fruits in the Sichuan Basin were often subjected to the threat of frost in the coldest months（from December to January next year）.The frost resulted in the great quantities of fruits going down and the yield down sharply.In the production,the widespread method was overlaied the ex-tree before the coldest month coming,and through this method could ease the fruit frost in some extent and hide the ray seriously.Thus it caused the crown of a tree to deteriorate badly in the light condition,and the fruit quality descended seriously.Currently,researchs on the citrus frost injury had focused on the leaves and branches, but researchs on the citrus fruit frost injury had not been reported,which was that the’ Skiranui tangerine’ fruit frost caused by the INA,and controlled method.In this study,’Skiranui tangerine’ hybrid citrus fruit was used,through separation and purification,it may be found to have INA bacteria of this fruit,and the INA were identificated.With the use of chemical reagents and the antagonistic bacterias which were isolated from Valencia Orange,blood orange and trifoliate orange,grapefruit and other fruit,the INA bacteria on the ’Skiranui tangerine’ fruit were prevented and cured in the room and in the field.We looked forward to find a way that may keep the quality of ’Skiranui tangerine’,and it was also safe and effective to control frost,and offered a reliable guarantee for high-quality’ Skiranui tangerine’ fruit.The main findings were as follows:（1） Separated by plate dilution and Vali small droplets freeze,9 strains of INA bacteria are isolated and identified from the surface of ’Skiranui tangerine’ fruit in the first time.INA 110（Erwinia ananas） and INA3023（Pseudomonas syringae pv.syringae） which are supplied by the molecule pathology group of key laboratory of plant diseases and insect pests biology of plant protection research institute of Institute of Agricultural Science in china is become standand strains.According to the bacteriology identification,4 isolatess arc Erwinia ananas and 5 isolates are Pseudomonas syringae pvs,respectively for 44.44% and 55.56%.（2） The activity of INA bacteria from ’Skiranui tangerine ’fruit is affected by temperature,bacteria mixture concentration and pH value.If the density of INA bacteria is 5×10²～5×10⁸ml^-1 and temperature is -2～-7℃,the density of INA bacteria is more higher and ice-nucleation activity is more better.When the density of INA bacteria is to some extent,temperature is more lower and ice-nucleation activity is more better.PH value of INA bacteria growth is 5.0～9.0,and the best pH value is 7.0,acidic solution（pH2～4）and alkaline solution（pH10） can destroy ice-nucleation activity of INA bacteria.Among three experimental medicaments as NO.1 frost-resistant medicament,lime-sulphate medicament and macozeb have good effect on controlling experiments,above 90%.NO.1 frost-resistant medicament（2000 times） shows the best effects on the INA bacteria.（3） In a simulated natural frost artificial box,ten kinds of chemical medicaments as NO.1 frost-resistant medicament（1000 times,1500 times,2000 times）,80%Macozeb（500 times）,72%Streptomycin（1000 times）,99.5%boric acid（200 times）,98%CuPFerron （1000 times）,98%ZNCl₂（1000 times）,and other chemical medicament that pH value of mixture which used Lime-sulphate medicament and boric acid is 4 and 10 are used to prevent and cure the INA bacteria.2000 times mixture of NO.1 frost-resistant medicament shows the best effects on controlling the INA bacteria in’ Skiranui tangerine’ fruit,and its killing rate is 99.80%.（4） 16 isolates of antagonistic bacteria of INA bacteria are isolated from ’Valencia Orange’ fruit and other four citrus fruits on the same ecological condition.Thereinto,D₁ bacteria isolated from ’Valencia Orange’ shows the best effects on controlling the INA bacteria indoor.（5） Under the low temperature condition（≤0℃,14d;frost day,Sd;snowy day,5d;the absolute minimum temperature is -5℃）,ten kinds of chemical medicaments as NO.1 frost-resistant medicament（1000 times,1500 times,2000 times）,80%Macozeb（500 times）,72%Streptomycin（1000 times）,99.5%Boric Acid（200 times）,98%CuPFerron（1000 times）,98%ZNCl₂（1000 times）,and other chemical medicament that pH value of mixture which used Lime-sulphate medicament and boric acid is 4 and 10 are used to prevent and cure in the field.NO.1 frost-resistant medicament（2000 times） and boric acid（200 times） show the better results,with both frostbite rates respectively are 8.90%and 10.00%.The effects on frost preventing in the field for ’Skiranui tangerine’ fruit of six antagonistic bacteria(D₁、D₂、D₅、D₇、D₉、D₁₂) which were isolated from ’Valencia Orange’ fruit and other four citrus fruits were not very ideal.But the effect of D₁,D₂ antagonistic bacterias from the ’Valencia Orange’（C.sinensis cv.Valencia orange） was better relatively.In six different approachs of physical frost preventing,as big house plastic sheeting, little house plastic sheeting,crown shallow coverage of plastic sheeting,fruit bagging （white）,fruit bagging（black inside and grey outside） and fruit bagging（brown paper）,big house plastic sheeting showed the best effect on preventing in the field,the frostbite rate is 5.57%,but the cost of big house plastic sheeting is high,so it is difficult to promote in the production.Above mentioned 22 different frost-resistant treatments as medicaments,biocontrol bacteriums、fruit baggings and equipment treatments and so on in the field,the equipment treatments had higher effect on the flower bud differentiation and the fruit quality,the single flower rate of having leaf is only 5.03%with crown shallow coverage of plastic sheeting.Synthetically considering the combination of factors,such as fruit quality,cost, operation and the output next year,the treatment of frost-resistant that used NO.1 frost-resistant medicament（2000 times）,boric acid（200 times） and fruit bagging（white） is good,and it can be widely used in the production.更多还原