JP2004109012A - Disease resistance evaluation method of plant and reproduction method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a disease resistance evaluation method of a plant for evaluating the plant having resistance against a pathogen highly accurately in a short time without requiring hard labor. <P>SOLUTION: This disease resistance evaluation method of the plant for testing resistance or sensibility of the test plant against a pathogen has a first step wherein the pathogen is inoculated into a sample of the test plant, and the sample of the test plant just after inoculation is irradiated with light, and generated delayed emission is detected as long as a prescribed time, a second step wherein the sample of the test plant into which the pathogen is inoculated is left as it is as long as a prescribed time under a prescribed condition, and then the sample of the test plant is irradiated with light, and the generated delayed emission is detected as long as a prescribed time, and a third step wherein the quantity of the delayed emission measured respectively in the first and second steps is compared each other to acquire a comparison value, to thereby evaluate the resistance or the sensibility of the test plant against the pathogen. <P>COPYRIGHT: (C)2004,JPO

Description

【００４０】
その結果、表２に示すように、クロマツ１３個体（クローン）の間で、枯れの程度に大きな差が認められた。１回目と２回目の試験で枯れの程度が同程度に現れる個体の中で、「津屋崎５０」、「土佐清水６３」、「三崎９０」などは枯れの程度が軽く、病原体であるセンチュウに対する抵抗性が高いと考えられ、また、「川内２９０」、「夜須３７」、「吉田２」などは枯れの程度が大きく、供試した１３個体の中では抵抗性が低いことが判明した。
【表２】

【００４１】
［病害抵抗性の評価（枝を試料に用いる方法）］
クロマツ個体（クローン）「津屋崎５０」、「三崎９０」、「川内２９０」及び「夜須３７」の当年枝を２つに分割し、針葉のついた上部を試験管内に水挿しし、マツノザイセンチュウを５０００頭接種した後、枯れの程度を表１の基準に基づいて調べた。
【００４２】
下部の切片については表面を削り、マツノザイセンチュウを２５００頭接種し、接種直後（接種６０分以内）に、図１に示す病害抵抗性測定装置と同様の構成を有する浜松ホトニクス社製ＡＲＧＵＳ５０／ＶＩＭシステムを用いて、３秒間の白色光を照射後、遅延発光（Ｄｅｌａｙｅｄ　Ｌｕｍｉｎｅｓｃｅｎｃｅ　：　ＤＬ）を３秒間測定した（得られた遅延発光の積算値をＤＬ_０とする。）。次いで、この試料を２４℃の薄暗い室内で７日間放置した。そして放置期間の毎日、すなわち、接種１日後、２日後、３日後、４日後、５日後、６日後及び７日後の遅延発光を上記と同様にして測定した（得られた遅延発光の積算値を、それぞれ、ＤＬ_１、ＤＬ_２、ＤＬ_３、ＤＬ_４、ＤＬ_５、ＤＬ_６及びＤＬ_７とする。）。そして、ＤＬ_１／ＤＬ_０、ＤＬ_２／ＤＬ_０、ＤＬ_３／ＤＬ_０、ＤＬ_４／ＤＬ_０、ＤＬ_５／ＤＬ_０、ＤＬ_６／ＤＬ_０、及びＤＬ_７／ＤＬ_０の値を比較値として求めた。かかる比較値を図２に示す。なお、図２においては、例えば「津屋崎５０」が２つ表されているが、これは母樹「津屋崎５０」の２ヶ所の枝についての結果を示すものである。また、図２にはそれぞれの個体について、枯れの程度が記載されているが、これは、試験管内に水挿しした上記当年枝の枯れの程度を表１の基準で評価したものである。
【００４３】
上記試験の対照試験として、２つに分割した「津屋崎５０」、「三崎９０」及び「川内２９０」、「夜須３７」の当年枝の下部の切片に、マツノザイセンチュウを接種せずに、蒸留水を処理した後、上記と同様にして遅延発光を測定した（得られた遅延発光の積算値をＤＬ’_０とする。）。そして、試験開始１日後、２日後、３日後、４日後、５日後、６日後及び７日後の遅延発光を上記と同様にして測定した（得られた遅延発光の積算値を、それぞれ、ＤＬ’_１、ＤＬ’_２、ＤＬ’_３、ＤＬ’_４、ＤＬ’_５、ＤＬ’_６及びＤＬ’_７とする。）。そして、ＤＬ’_１／ＤＬ’_０、ＤＬ’_２／ＤＬ’_０、ＤＬ’_３／ＤＬ’_０、ＤＬ’_４／ＤＬ’_０、ＤＬ’_５／ＤＬ’_０、ＤＬ’_６／ＤＬ’_０、及びＤＬ’_７／ＤＬ’_０の値を比較値として求めた。かかる比較値を図３に示す。なお、図３においては、例えば「津屋崎５０」が２つ表されているが、これは母樹「津屋崎５０」の２ヶ所の枝についての結果を示すものである。
【００４４】
図３に示されるように、枯れの程度が大きく異なる試料であるにもかかわらず、蒸留水処理のみでは、ＤＬ値の比（ＤＬ値／処理直後のＤＬ値）は、供試個体（クローン）の間で経時的に大きな違いは認められなかった。
【００４５】
これに対し、マツノザイセンチュウを接種した試料については、ＤＬ値の比（ＤＬ値／接種直後のＤＬ値）は日数の経過とともに個体間の差が大きくなり、分散分析の結果、接種から７日後に１％水準で個体間に有意差が認められた（図２）。また、マツノザイセンチュウ接種試験で枯れの程度が大きいものほどＤＬ値の比（ＤＬ値／接種直後のＤＬ値）が小さくなる傾向にあり、接種から７日後の遅延発光の比と枯れの程度との相関係数はｒ＝−０．８３７＊であった。
【００４６】
これらの結果から、マツノザイセンチュウに対して抵抗性を持つ個体と感受性の個体を、当年枝切片の遅延発光を測定することにより識別が可能であることがわかった。特に、ＤＬ値の比（ＤＬ値／接種直後のＤＬ値）があまり変化しない個体が存在し、これらは病害抵抗性が高い個体であることがわかった。一方、ＤＬ値の比（ＤＬ値／接種直後のＤＬ値）が大きく低下する個体が存在し、これらは病害抵抗性が低い個体であることがわかった。
【００４７】
マツノザイセンチュウ接種試験による枯れの程度と、マツノザイセンチュウ接種７日後の、ＤＬ値の比（初期値に対する遅延発光の比）をまとめて表３に示した。
【表３】

【００４８】
なお、病害抵抗性（枝）の評価において、遅延発光の波長は６５０ｎｍ〜８００ｎｍの範囲にあり、特に６８０ｎｍ〜７００ｎｍと７３０ｎｍ前後にピークが認められた。
【００４９】
［病害抵抗性の評価（葉を試料に用いる方法）］
「津屋崎５０」、「三崎９０」、「川内２９０」、「夜須３７」、「波方７３ａ」及び「吉田２ａ」の当年枝を試験管内に水挿しし、マツノザイセンチュウ５０００頭を接種した後の枯れの程度を表１の基準に基づいて調べた。なお、本発明において「津屋崎５０」のように地名の後に数字を付した個体名は、接木により得られたクローンを示し、「波方７３ａ」のように末尾にａを付した個体名は、接木により得られたクローンである「波方７３」から得られた種子を育成して得られた個体を意味する。
【００５０】
一方、「津屋崎５０」、「三崎９０」、「川内２９０」、「夜須３７」、「波方７３ａ」及び「吉田２ａ」の当年枝から採取した針葉を３ｃｍの長さに切断し、１０００００頭／３ｍＬのマツノザイセンチュウ懸濁液に２０時間浸漬した。浸漬直後（浸漬後６０分以内）に、１個体につき１２本の針葉をシャーレに並べて、浜松ホトニクス社製ＡＲＧＵＳ５０／ＶＩＭシステムを用いて、３秒間の白色光を照射後、遅延発光を３秒間測定した（得られた遅延発光の積算値をＤＬ_１０とする。）。
【００５１】
次いで、この試料を２４℃の薄暗い室内で１２日間放置した。そして、接種４日後、６日後、８日後、１０日後及び１２日後の遅延発光を上記と同様にして測定した（得られた遅延発光の積算値を、それぞれ、ＤＬ_１１、ＤＬ_１２、ＤＬ_１３、ＤＬ_１４及びＤＬ_１５とする。）。そして、ＤＬ_１１／ＤＬ_１０、ＤＬ_１２／ＤＬ_１０、ＤＬ_１３／ＤＬ_１０、ＤＬ_１４／ＤＬ_１０、及びＤＬ_１５／ＤＬ_１０の値を比較値として求めた。かかる比較値を図４に示す。
【００５２】
上記試験の対照試験として、「津屋崎５０」、「三崎９０」、「川内２９０」、「夜須３７」、「波方７３ａ」及び「吉田２ａ」の当年枝について、蒸留水に浸漬した後に上記と同様にして遅延発光を測定した（得られた遅延発光の積算値をＤＬ’_１０とする。）。そして、試験開始４日後、６日後、８日後、１０日後及び１２日後の遅延発光を上記と同様にして測定した（得られた遅延発光の積算値を、それぞれ、ＤＬ’_１１、ＤＬ’_１２、ＤＬ’_１３、ＤＬ’_１４及びＤＬ’_１５とする。）。そして、ＤＬ’_１１／ＤＬ’_１０、ＤＬ’_１２／ＤＬ’_１０、ＤＬ’_１３／ＤＬ’_１０、ＤＬ’_１４／ＤＬ’_１０、及びＤＬ’_１５／ＤＬ’_１０の値を比較値として求めた。かかる比較値を図５に示す。
【００５３】
図５に示されるように、蒸留水処理のみでは、ＤＬ値の比（ＤＬ値／処理直後のＤＬ値）は、供試個体（クローン）の間で経時的に大きな違いは認められなかったが、マツノザイセンチュウを接種した試料については、ＤＬ値の比（ＤＬ値／接種直後のＤＬ値）は日数の経過とともに個体間の差が大きくなり（図４）、分散分析の結果、接種から１２日後では１％水準で個体間に有意差が認められ、遅延発光の比と枯れの程度との相関係数はｒ＝−０．７３０であった。
【００５４】
これらの結果から、マツノザイセンチュウ病に対して抵抗性を持つ個体と感受性の個体を、針葉の遅延発光を測定することにより識別が可能であることがわかった。
【００５５】
［枝の褐変の程度と遅延発光との関係］
上記「病害抵抗性の評価（枝を試料に用いる方法）」において、接種１日後、２日後、３日後、４日後、５日後、６日後及び７日後に、ＤＬ_１／ＤＬ_０、ＤＬ_２／ＤＬ_０、ＤＬ_３／ＤＬ_０、ＤＬ_４／ＤＬ_０、ＤＬ_５／ＤＬ_０、ＤＬ_６／ＤＬ_０、及びＤＬ_７／ＤＬ_０の値を求めると共に、接種１日後、２日後、３日後、４日後、５日後、６日後及び７日後における褐変の程度を、以下の表４に基づいて評価した。そして、ＤＬ値の比（ＤＬ値／接種直後のＤＬ値）と褐変の程度との関係を評価した。
【表４】

【００５６】
その結果、マツノザイセンチュウ接種から７日後におけるＤＬ値の比（ＤＬ値／接種直後のＤＬ値）と褐変の程度との相関係数はｒ＝−０．９５６＊＊であった。したがって、接種７日後のＤＬ値の比（ＤＬ値／接種直後のＤＬ値）を用いることにより、枝の褐変の程度を評価可能であることがわかった。
【００５７】
［針葉の褐変の程度と遅延発光との関係］
上記「病害抵抗性の評価（葉を試料に用いる方法）」において、接種４日後、６日後、８日後、１０日後及び１２日後に、ＤＬ_１１／ＤＬ_１０、ＤＬ_１２／ＤＬ_１０、ＤＬ_１３／ＤＬ_１０、ＤＬ_１４／ＤＬ_１０及びＤＬ_１５／ＤＬ_１０の値を求めると共に、接種４日後、６日後、８日後、１０日後及び１２日後における褐変の程度を、上記表４に基づいて評価した。そして、ＤＬ値の比（ＤＬ値／接種直後のＤＬ値）と褐変の程度との関係を検討した。
【００５８】
その結果、マツノザイセンチュウ接種から１２日後におけるＤＬ値の比（ＤＬ値／接種直後のＤＬ値）と褐変の程度との相関係数はｒ＝−０．８７６＊であった。したがって、接種１２日後のＤＬ値の比（ＤＬ値／接種直後のＤＬ値）を用いることにより、針葉の褐変の程度を評価可能であることがわかった。
【００５９】
［植物の病害抵抗性評価方法における照射光の影響］
「津屋崎５０」、「三崎９０」、「川内２９０」及び「夜須３７」の当年枝を試験管内に水挿しし、マツノザイセンチュウ５０００頭を接種した後の枯れの程度を表１の基準に基づいて調べた。
【００６０】
一方、「津屋崎５０」、「三崎９０」、「川内２９０」、「夜須３７」の当年枝から採取した針葉を３ｃｍの長さに切断し、表面を薄く削り取った後に、１０００００頭／３ｍＬのマツノザイセンチュウ懸濁液に２０時間浸漬した。１個体につき１０本の針葉をシャーレに並べて、２４℃の薄暗い室内で６日間放置した。
【００６１】
次いで、この試料を浜松ホトニクス社製ＡＲＧＵＳ５０／ＶＩＭシステムを用いて、発光ダイオードから発する青色光（中心波長４７０ｎｍ）、緑色光（中心波長５５０ｎｍ）又は赤色光（中心波長６５０ｎｍ）をそれぞれ５秒間照射後、遅延発光を０．１秒間測定した（青色光、緑色光及び赤色光により得られた遅延発光の積算値を、それぞれＤＬ_ｂ６、ＤＬ_ｇ６及びＤＬ_ｒ６とする。）。それぞれの光によるＤＬ値を図６に示す。また、接種６日後の枯れの程度を表１の基準で求めた。
【００６２】
そして、ＤＬ値と枯れの程度との相関を、青色光、緑色光又は赤色光それぞれについて求めたところ、相関係数は赤色光でｒ＝−０．６８６、青色光でｒ＝−０．６１０、緑色光でｒ＝−０．０８４であった。この結果から、葉を用いてマツノザイセンチュウに対する抵抗性を評価するにあたっては、照射光として、赤色光及び青色光が特に有効であることが判明した。
【００６３】
さらに、赤色光によるＤＬ値／緑色光によるＤＬ値、青色光によるＤＬ値／緑色光によるＤＬ値と枯れの程度との相関を求めたところ、相関係数は赤色光によるＤＬ値／緑色光によるＤＬ値でｒ＝−０．７０９＊、青色光によるＤＬ値／緑色光によるＤＬ値でｒ＝−０．６７０であった（図７）。この結果から、葉を用いてマツノザイセンチュウに対する抵抗性を評価するにあたっては、照射光として、赤色光、青色光及び緑色光が有効であり、これらの光によるＤＬ値をそれぞれ組み合わせることによって評価の精度がさらに高まることが判明した。
【００６４】
【発明の効果】
以上説明したように、本発明によれば、病原体に対して抵抗性（又は感受性）のある植物を、重労働を必要とせずに短期間に高い精度で評価する植物の病害抵抗性評価方法を提供することが可能になる。
【図面の簡単な説明】
【図１】本発明において用いることのできる病害抵抗性測定装置の概略図である。
【図２】マツノザイセンチュウを接種したクロマツ（枝）の遅延発光量の比の経時変化を示す図である。
【図３】対照として蒸留水処理したクロマツ（枝）の遅延発光量の比の経時変化を示す図である。
【図４】マツノザイセンチュウを接種したクロマツ（針葉）の遅延発光量の比の経時変化を示す図である。
【図５】対照として蒸留水処理したクロマツ（針葉）の遅延発光量の比の経時変化を示す図である。
【図６】照射光の種類の違いによる遅延発光量を示す図である。
【図７】赤色光又は青色光の照射によるＤＬ値を緑色光の照射によるＤＬ値で除した場合の比を示す図である。
【符号の説明】
２・・・病害抵抗性測定装置、４・・・遅延発光測定装置、６・・・制御部、７・・・出力部、８・・・被検植物試料、１０・・・暗箱、１１・・・投光系、１２・・・ＶＩＭカメラ、１６・・・光出射部、３０・・・試料設置台、３２・・・レンズ、３３・・・フィルタ、３４・・・光源、３６・・・光ファイバ、３８・・・シャッター、３９・・・シャッターコントローラ、４０・・・イメージインテンシファイア、４２・・・ＣＣＤカメラ、５０・・・ＶＩＭカメラコントローラ、５２・・・イメージプロセッサ、５４・・・画像出力モニタ、５６・・・データ解析装置、５８・・・データ出力モニタ[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for evaluating plant disease resistance.
[0002]
[Prior art]
The damage of pine wilt caused by pine wood nematode disease has been recognized nationwide except in Hokkaido and Aomori Prefecture, and the amount of damaged wood has increased in recent years due to high temperatures in summer. In recent years, from the viewpoint of environmental problems, it has been refrained from aerial spraying of drugs that suppress pine wood nematodes, which mediate pine wood nematodes, and as an alternative, breeding pine seedlings that are resistant to pine wood nematodes, The idea of planting ash in a withered area has become widespread.
[0003]
Since pine is a tree species that is difficult to grow by cuttings, the following test was required prior to the planting. In other words, individuals that survived in the severely damaged area of pine wilt were first propagated by grafting, and those that had survived by inoculating pine wood nematodes on the proliferated pine were selected and used as seed trees in the seed orchard as mother trees. , And seedlings were obtained from the seeds obtained therefrom, and pine wood nematodes were inoculated on the seedlings to perform the test.
[0004]
[Problems to be solved by the invention]
However, this method has a drawback that, in addition to heavy labor performed in summer, the test results are greatly affected by weather conditions, and it takes a long time to obtain the results. As a result, the price of the seedlings subjected to the above-mentioned test was higher than that of general pine. Also, since the tree itself is inoculated with the nematode, it was impossible to test individuals (mother tree, natural monument, etc.) that could not be killed.
[0005]
The present invention has been made in view of the problems of the prior art, and is a method for evaluating plant disease resistance that evaluates plants that are resistant (or susceptible) to a pathogen. An object of the present invention is to provide a method that can evaluate plant disease resistance with high accuracy in a short period of time without using heavy labor, using some tissues. Another object of the present invention is to provide a method for growing a plant having resistance to a pathogen by using a plant selected by the method for evaluating disease resistance.
[0006]
[Means for Solving the Problems]
The present inventors have conducted intensive studies to solve the above problems, and as a result, the integrated value of the delayed luminescence generated in a certain period of time by irradiating the plant immediately after inoculation of the pathogen, and a certain period after the inoculation of the pathogen The present inventors have found that the above object can be achieved by comparing the integrated value of the delayed luminescence generated in a certain period of time by irradiating the plant with light, and completed the present invention.
[0007]
That is, the present invention relates to a method for evaluating the disease resistance of a plant for evaluating the resistance or sensitivity of a test plant to a pathogen, comprising: (1) inoculating the sample of the test plant with the pathogen; A first step of irradiating the sample of the plant with light to detect the delayed luminescence generated for a predetermined time; and (2) leaving the sample of the test plant inoculated with the pathogen under a predetermined condition for a predetermined time. A second step of irradiating the sample of the test plant with the light and detecting a delayed emission generated for a predetermined time; and (3) an amount of the delayed emission measured in the first and second steps, respectively. A third step of evaluating the resistance or susceptibility of the test plant to the pathogen by determining a comparison value by comparing the disease resistance of the test plant with the test plant. so That.
[0008]
As described above, since the present invention evaluates plants that are resistant or susceptible to a pathogen using delayed luminescence, objective and highly accurate evaluation is possible. In addition, since a sample obtained from the test plant instead of the test plant itself is used, the evaluation does not become large, and it is not necessary to perform the evaluation outdoors and in the summer season, so that heavy labor is not required. Furthermore, when a plant itself is used as a subject, resistance or the like can be evaluated only after a certain period of time has elapsed, whereas the method of the present invention described above requires a short period (typically several days). (Several weeks to several weeks), so that plants that are resistant or susceptible to the pathogen can be quickly selected.
[0009]
Preferably, the comparison value is a value obtained by dividing the amount of the delayed light emission detected in the second step for the predetermined time by the amount of the delayed light emission detected in the first step for the predetermined time. By using such a value, it is possible to objectively evaluate disease resistance with higher accuracy.
[0010]
In the disease resistance evaluation method of the present invention, it is preferable to use white light, red light, blue light or green light as the light. By using such light, it becomes possible to evaluate disease resistance with high accuracy. In addition, by combining the delayed emission values obtained from these lights, it becomes possible to evaluate disease resistance with higher accuracy.
[0011]
When the disease resistance evaluation method of the present invention is applied, the test plant sample may be a branch or a leaf of the test plant. By using a branch or a leaf as a sample, it is possible to judge resistance or the like based on delayed emission in a short period of time.
[0012]
The present invention also provides (1) inoculating each of two or more test plant samples with a pathogen, irradiating the test plant sample immediately after the inoculation with light, and generating the delayed luminescence for a predetermined time, respectively. A first step of detecting, and (2) irradiating each of the test plant samples with the light after leaving the test plant sample inoculated with the pathogen under a predetermined condition for a predetermined time. (2) detecting the amount of the delayed light emission detected in the second step for the predetermined time in the first step; A third step of obtaining a value obtained by dividing by the amount of the delayed luminescence detected for the predetermined time, and (4) a test plant showing a value lower than the average of the above values, which is significantly analyzed by analysis of variance of the above values. The test plants showing the appropriate values A fourth step of selecting only test plants having the resistance to the pathogen by excluding the test plants from the above test plants, and (5) sexually propagating or excluding the test plants selected in the fourth step. A fifth step of sexually breeding to obtain a plant having resistance to a pathogen. By carrying out such a propagation method, a next-generation plant or the like having resistance to a pathogen can be easily produced.
[0013]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, an embodiment of the method for evaluating plant disease resistance according to the present invention will be described step by step.
[0014]
First, the first step will be described. In the first step, the pathogen is inoculated into a sample of the test plant, and the sample of the test plant immediately after the inoculation is irradiated with light to detect the generated delayed luminescence for a predetermined time.
[0015]
Although the type of the test plant used in the present invention is arbitrary, it is preferable to use pine, and in particular, it is preferable to use pine, which is severely damaged by pine wilt. On the other hand, examples of the pathogen inoculated to the test plant include fungi, bacteria, viruses, and parasites. Parasites are preferable, and pine wood nematodes are particularly preferable. Therefore, the method for evaluating disease resistance of the present invention is optimally applied as a method for evaluating the resistance of pine to pine wood nematodes.
[0016]
In inoculating the test plant with the pathogen, a part of the test plant is used as a sample. In the present invention, the sample of the test plant is preferably a branch or a leaf from the viewpoint of ease of measurement and ease of judgment by delayed luminescence. As a method of inoculating such a sample with a pathogen, any known method can be adopted. For example, when using a branch or a leaf of a test plant as a sample, it is preferable that the surface thereof is shaved thinly and that the pathogen is directly inoculated or immersed in a solution containing the pathogen.
[0017]
In the first step, a sample immediately after inoculation of the pathogen is used to measure the initial value of delayed luminescence of the test plant. Here, "immediately after inoculation" means within 3 hours (preferably within 1 hour) after inoculation.
[0018]
The light applied to the sample of the test plant immediately after inoculation is preferably white light emitted from a xenon lamp light source or the like, or red light, blue light or green light emitted from a light emitting diode light source or the like. It is preferable to irradiate the irradiated surface and detect the delayed light emission generated from this surface. The light irradiation time is preferably 2 to 5 seconds (preferably 3 seconds in the case of a xenon lamp light source; preferably 5 seconds in the case of a light emitting diode light source), and the delayed light emission detection time is 0.1 to 4 seconds. (Preferably 3 seconds for a xenon lamp light source; preferably 0.1 seconds for a light emitting diode light source).
[0019]
FIG. 1 schematically shows a disease resistance measuring device 2 applied to the evaluation of disease resistance and measuring delayed luminescence emitted from a test plant. The disease resistance measurement device 2 mainly includes a delayed luminescence measurement device 4 that captures delayed luminescence from the test plant sample 8, a control unit 6 that performs appropriate processing on the captured data, and an output unit that outputs and displays a captured image and the like. 7.
[0020]
The delayed luminescence measuring device 4 is provided with a dark box 10 having a structure capable of blocking external light, and a sample setting table 30 in which the test plant sample 8 can be set is provided in the dark box 10. . The delayed luminescence measuring device 4 has a light projecting system 11 for irradiating the test plant sample 8 with light, and the light projecting system 11 can shield light from the light source 34 that emits light and light from the light source 34. A shutter 38 and two light emitting portions 16 connected to the shutter 38 via the optical fiber 36.
[0021]
The light emitting units 16 are provided above the sample setting table 30 in the dark box 10, and each light emitting unit 16 irradiates light from the light source 34 to the test plant sample 8. The shutter 38 disposed between the light source 34 and the light emitting unit 16 can shield light from the light source 34 under the control of the shutter controller 39, and the shutter 38 and the shutter controller 39 cooperate with each other. Light irradiation can be performed on the test plant sample 8 for a desired time.
[0022]
The irradiation light from the light source 34 can be, for example, white light. In this case, a xenon lamp can be used as the light source 34. The intensity at the irradiation position of the test plant sample 8 is 0.1 to 10 mW / cm. ² It is preferable that The light source 34 may be a blue light-emitting diode, a green light-emitting diode, or a red light-emitting diode. In such a case, the blue light, the green light, or the red light emits 0.1 to 10 mW at the irradiation position of the test plant sample 8. / Cm ² Irradiation is preferably performed so that
[0023]
A VIM camera 12 provided with a filter 33, a lens 32, an image intensifier 40, and a CCD camera 42 in this order from the sample setting table 30 side is installed in the upper part of the dark box 10. In the delayed emission from the test plant sample 8, only a specific wavelength component passes through the filter 33, and the passing light is collected by the lens 32. Further, the collected delayed emission is multiplied after being photoelectrically converted by a photocathode provided before the image intensifier 40, and the multiplied electrons collide with the subsequent phosphor screen to form a fluorescent pattern. It is formed. Then, the fluorescent pattern formed on the fluorescent screen is imaged by the CCD camera 42. Since the VIM camera 12 according to the present embodiment has such a configuration, it is possible to detect even extremely weak light emission.
[0024]
The control unit 6 includes a VIM camera controller 50 for controlling the voltage of the VIM camera 12, an image processor 52 for processing an image signal obtained by the VIM camera 12, and a data analyzer 56 for quantifying the obtained image data. are doing. The output unit 7 has an image output monitor 54 for outputting and displaying the image data processed by the image processor 52 and a data output monitor 58 for outputting and displaying the quantified data. The data analyzer 56 is connected to the VIM camera controller 50, the image processor 52, and the above-described shutter controller 39, and can perform each control.
[0025]
The image signal obtained by the VIM camera 12 is sent to the VIM camera controller 50, which adjusts the sensitivity of the VIM camera 12 by controlling the voltages of the image intensifier 40 and the CCD camera 42. . Further, the VIM camera controller 50 also has a function of preventing a failure of the VIM camera 12. That is, since the VIM camera 12 detects the delayed light emission under the condition of a small amount of light, there is a possibility that the VIM camera 12 may break down due to the entry of noise light during operation. Therefore, the VIM camera controller 50 connected to the dark box 10 monitors the opening / closing state of the door of the dark box 10 while the VIM camera 12 is operating, and cuts off the voltage of the VIM camera 12 when detecting the opening operation of the door. ing. In this manner, the VIM camera controller 50 prevents the failure of the VIM camera 12 due to the entry of noise light.
[0026]
Further, the image signal sent from the VIM camera controller 50 is subjected to image processing by the image processor 52, and the image captured by the CCD camera 42 is displayed on the image output monitor 54. The image data processed by the image processor 52 is quantified by the data analyzer 56 as the amount of delayed light emission, and the quantified data is output to the data output monitor 58. Thereby, the operator can visually recognize the delayed light emission amount of the test plant sample 8 and can evaluate the degree of disease resistance of the sample based on the delayed light emission amount. The data analyzer 56 may not only calculate the amount of delayed luminescence from the test plant sample 8 but also calculate a comparison value with the amount of delayed luminescence in another sample. The degree of disease resistance of the sample may be evaluated by comparing the comparison value with a predetermined reference value.
[0027]
Next, the second step of the method for evaluating plant disease resistance according to the present invention will be described. In the second step, the sample of the test plant inoculated with the pathogen is left under a predetermined condition for a predetermined time, and then the sample of the test plant is irradiated with light, and the generated delayed luminescence is detected for a predetermined time.
[0028]
The condition for leaving the sample inoculated with the pathogen in the second step is appropriately determined depending on the type of the test plant to be evaluated, the site of the test plant from which the sample is obtained, and the like. For example, when using a branch or a leaf of a black pine as a sample, it is preferable to leave it in a dark room at 24 ° C., and as a leaving time, 5 to 9 days (preferably 6 to 8 days, more preferably Is 7 days), and in the case of leaves, 10 to 14 days (preferably 11 to 13 days, more preferably 12 days). Then, under the same conditions (irradiation time, detection time, irradiation intensity, etc.) as those in the first step, it is preferable to measure the delayed light emission on the same surface as the surface irradiated with light in the first step.
[0029]
Next, the third step of the method for evaluating plant disease resistance according to the present invention will be described. In the third step, the amount of delayed emission measured in the first step (DL _x ) And the amount of delayed emission measured in the second step (DL _y ) To evaluate the resistance or susceptibility of the test plant to the pathogen.
[0030]
The comparison of the delayed light emission amount is, for example, DL _x And DL _y Difference or DL _x And DL _y Can be used, and in the present invention, DL _y / DL _x It is preferable to use
[0031]
According to the findings of the present inventors, the test plant is DL according to its type. _y / DL _x Changes significantly with time, and DL _y / DL _x Are divided into those with little change over time. Where DL _y / DL _x Changes over time are thought to correspond to individual changes due to pathogens. _y / DL _x Can be identified as those with little change over time as plants with high resistance, and those with greatly reduced as plants with low resistance to pathogens. DL _y / DL _x In determining whether or not there is resistance to a pathogen based on the above, for example, the following criteria can be adopted. That is, the DL of each test plant _y / DL _x Is analyzed, and a significant test plant is selected at the 5% level (1% level when stricter). DL of the selected test plants _y / DL _x Are selected as test plants having a lower value than the average value of the whole, and are selected as test plants having low resistance to pathogens. If a significant test plant cannot be selected based on such criteria, the time left in the second step may be extended until a significant value appears in the analysis of variance.
[0032]
For example, when black pine is used as a test plant and pine wood nematode is used as a pathogen, about 7 days after pathogen inoculation when the sample is a branch, and about 12 days after pathogen inoculation when the sample is a leaf. , Low pine resistance to pine wood nematode DL _y / DL _x Significantly decreases. And DL _y / DL _x There is a high correlation between the decrease in the value of pine and the withering and browning of black pine. Therefore, DL _y / DL _x Is evaluated for a short period of time of 7 days or 12 days, whereby the resistance (or susceptibility) to the pathogen can be evaluated with high accuracy. Therefore, as compared with the conventional method of placing the test plant itself outdoors for evaluation, not only the evaluation period is significantly shortened, but also the objectivity of the evaluation is improved and the required labor is minimized. be able to.
[0033]
Next, an embodiment of the method for growing a plant according to the present invention will be described step by step. First, in the first step, two or more test plants are prepared as test plants. Here, the test plants are preferably selected from plants of the same species. For example, when growing pine trees having resistance to pathogens, it is preferable to use only pine trees as test plants. In the first step, delayed luminescence can be detected using the same device as the disease resistance measuring device 2 that can be used in the above-described disease resistance evaluation method, and suitable types of light and test plants And the pathogen is the same as in the above-mentioned disease resistance evaluation method.
[0034]
The second step can be performed in the same manner as the second step of the above-described disease resistance evaluation method. In the third step, similarly to the above, the delayed light emission detected for a predetermined time in the second step is performed. A value is obtained by dividing the amount by the amount of delayed light emission detected for a predetermined time in the first step.
[0035]
In the fourth step, a variance analysis of the values obtained in the third step is performed to select test plants having resistance to the pathogen. Also in this case, the same criterion can be adopted. That is, the DL of each test plant _y / DL _x Is analyzed for variance, and for example, a significant test plant is selected at the 5% level (or 1% level when stricter). DL of the selected test plants _y / DL _x Are selected as test plants having a lower value than the average value of the whole, and are selected as test plants having low resistance to pathogens. If a significant test plant cannot be selected based on such criteria, the time left in the second step may be extended until a significant value appears in the analysis of variance.
[0036]
In the fifth step, the test plants selected in the fourth step are sexually or asexually propagated to obtain plants having resistance to the pathogen. Here, sexual reproduction includes mating, and asexual reproduction includes strain division, cuttings, grafting, tissue culture, genetic manipulation, and the like. By carrying out the fifth step, it is possible to produce and raise a next-generation plant individual or the like having excellent resistance to diseases. Further, if necessary, these next-generation plants and the like are evaluated by the disease resistance evaluation method of the present invention, and it is possible to more reliably produce and raise seedlings having excellent disease resistance.
[0037]
【Example】
Hereinafter, preferred embodiments of the present invention will be described in more detail, but the present invention is not limited to these embodiments.
[0038]
[Pine selection for evaluation]
To examine whether the disease resistance evaluation method of the present invention can be applied for evaluating the resistance or susceptibility of a plant to a pathogen, a test plant having a high resistance to a pathogen and a resistance to a pathogen are originally used. Results need to be compared using lower test plants. Therefore, first, a test for selecting a test plant having a high resistance to a pathogen and a test plant having a low resistance was carried out as follows.
[0039]
First, this year's branch of Japanese black pine (clone), which is considered to be resistant to pine wood nematodes selected from pine wilt severely damaged areas in southwestern Japan, was watered into test tubes. The black pine used was "Tsuyazaki 50", "Tosa Shimizu 63", "Misaki 90", "Namikata 73", "Shima 64", "Miyoyo 103", "Oseto 12", "Namikata 37", "Ei" 425 "," Tanabe 54 "," Yoshida 2 "," Yasu 37 "and" Kawauchi 290 ". 5000 nematodes were inoculated to these Japanese black pine, and the degree of withering was investigated in accordance with the criteria shown in Table 1.
[Table 1]

[0040]
As a result, as shown in Table 2, a great difference was observed in the degree of withering among the 13 black pine individuals (clones). Among the individuals showing the same degree of withering in the first and second tests, "Tsuyazaki 50", "Tosashimizu 63", "Misaki 90", etc. have a low degree of withering and resistance to the pathogen, nematode It is considered that the mortality is high, and "Kawauchi 290", "Yasu 37", "Yoshida 2", etc. have a high degree of withering, and it was found that the 13 individuals tested had low resistance.
[Table 2]

[0041]
[Evaluation of disease resistance (method using branch as sample)]
Japanese black pine individuals (clone) “Tsuyazaki 50”, “Misaki 90”, “Kawauchi 290” and “Yasu 37” were split into two, and the upper part with needles was watered into a test tube. After inoculating 5000 nematodes, the degree of withering was examined based on the criteria in Table 1.
[0042]
For the lower section, the surface was shaved, and 2500 pine wood nematodes were inoculated. Immediately after inoculation (within 60 minutes of inoculation), ARGUS50 / VIM manufactured by Hamamatsu Photonics Co., Ltd. having the same configuration as the disease resistance measurement device shown in FIG. After irradiating white light for 3 seconds using the system, delayed luminescence (DL) was measured for 3 seconds (the integrated value of the obtained delayed luminescence was DL). ₀ And ). The sample was then left in a dark room at 24 ° C. for 7 days. Then, the delayed luminescence was measured in the same manner as described above every day of the standing period, that is, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and 7 days after inoculation (the integrated value of the obtained delayed luminescence was measured in the same manner as described above). , Respectively, DL ₁ , DL ₂ , DL ₃ , DL ₄ , DL ₅ , DL ₆ And DL ₇ And ). And DL ₁ / DL ₀ , DL ₂ / DL ₀ , DL ₃ / DL ₀ , DL ₄ / DL ₀ , DL ₅ / DL ₀ , DL ₆ / DL ₀ , And DL ₇ / DL ₀ Was determined as a comparative value. FIG. 2 shows such comparison values. In FIG. 2, for example, two “Tsuyazaki 50” are shown, but this shows the result for two branches of the mother tree “Tsuyazaki 50”. FIG. 2 shows the degree of withering for each individual. This is obtained by evaluating the degree of withering of the current year branch watered in a test tube with reference to Table 1.
[0043]
As a control test of the above test, distillation was performed without inoculating pine wood nematode on the lower section of the current branch of “Tsuyazaki 50”, “Misaki 90”, “Kawauchi 290”, and “Yasu 37” divided into two. After treating the water, delayed luminescence was measured in the same manner as described above (the integrated value of the obtained delayed luminescence was DL ′). ₀ And ). Then, delayed light emission was measured in the same manner as described above 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and 7 days after the start of the test (the integrated values of the obtained delayed luminescence were DL ′, respectively). ₁ , DL ' ₂ , DL ' ₃ , DL ' ₄ , DL ' ₅ , DL ' ₆ And DL ' ₇ And ). And DL ' ₁ / DL ' ₀ , DL ' ₂ / DL ' ₀ , DL ' ₃ / DL ' ₀ , DL ' ₄ / DL ' ₀ , DL ' ₅ / DL ' ₀ , DL ' ₆ / DL ' ₀ , And DL ' ₇ / DL ' ₀ Was determined as a comparative value. FIG. 3 shows such comparison values. In FIG. 3, for example, two “Tsuyazaki 50” are shown, but this shows the results for two branches of the mother tree “Tsuyazaki 50”.
[0044]
As shown in FIG. 3, the ratio of the DL value (DL value / DL value immediately after the treatment) was not improved by the distilled water treatment alone, even though the samples had greatly different degrees of withering. No significant difference was observed over time between the two.
[0045]
On the other hand, for the sample inoculated with the pine wood nematode, the ratio of the DL value (DL value / DL value immediately after the inoculation) became larger among individuals with the passage of days, and as a result of analysis of variance, 7 days after the inoculation. Later, a significant difference was observed between individuals at the 1% level (FIG. 2). Moreover, the ratio of DL value (DL value / DL value immediately after inoculation) tends to decrease as the degree of withering increases in the pine wood nematode inoculation test. Was r = -0.837 *.
[0046]
From these results, it was found that individuals having susceptibility to pine wood nematodes and those susceptible could be distinguished by measuring delayed luminescence of current year branch sections. In particular, there were individuals in which the ratio of DL values (DL value / DL value immediately after inoculation) did not change much, and it was found that these individuals had high disease resistance. On the other hand, there were individuals in which the ratio of DL values (DL value / DL value immediately after inoculation) significantly decreased, and it was found that these individuals had low disease resistance.
[0047]
Table 3 summarizes the degree of withering in the pine wood nematode inoculation test and the ratio of the DL value (the ratio of the delayed luminescence to the initial value) 7 days after the pine wood nematode inoculation.
[Table 3]

[0048]
In the evaluation of disease resistance (branches), the wavelength of delayed light emission was in the range of 650 nm to 800 nm, and particularly peaks were observed at 680 nm to 700 nm and around 730 nm.
[0049]
[Evaluation of disease resistance (method using leaves as a sample)]
After inoculating the current branch of "Tsuyazaki 50", "Misaki 90", "Kawauchi 290", "Yasu 37", "Namikata 73a" and "Yoshida 2a" into a test tube, inoculate 5,000 pine wood nematodes The degree of withering was examined based on the criteria in Table 1. In the present invention, an individual name with a number added after a place name such as “Tsuyazaki 50” indicates a clone obtained by grafting, and an individual name with a suffix a such as “Namikata 73a” is It means an individual obtained by growing seeds obtained from “Namikata 73” which is a clone obtained by grafting.
[0050]
On the other hand, needles collected from the current branch of "Tsuyazaki 50", "Misaki 90", "Kawauchi 290", "Yasu 37", "Namikata 73a" and "Yoshida 2a" were cut into 3 cm lengths and cut into 100,000 pieces. The head was immersed in a pine wood nematode suspension of 3 mL for 20 hours. Immediately after immersion (within 60 minutes after immersion), 12 needles per individual are arranged in a petri dish, irradiated with white light for 3 seconds using an ARGUS50 / VIM system manufactured by Hamamatsu Photonics, and delayed emission is performed for 3 seconds. Measured (the integrated value of the obtained delayed light emission is DL ₁₀ And ).
[0051]
The sample was then left in a dark room at 24 ° C. for 12 days. After 4 days, 6 days, 8 days, 10 days, and 12 days after the inoculation, the delayed luminescence was measured in the same manner as described above (the integrated value of the obtained delayed luminescence was DL, respectively). ₁₁ , DL ₁₂ , DL _Thirteen , DL ₁₄ And DL _Fifteen And ). And DL ₁₁ / DL ₁₀ , DL ₁₂ / DL ₁₀ , DL _Thirteen / DL ₁₀ , DL ₁₄ / DL ₁₀ , And DL _Fifteen / DL ₁₀ Was determined as a comparative value. FIG. 4 shows such comparison values.
[0052]
As a control test of the above test, the current branches of "Tsuyazaki 50", "Misaki 90", "Kawauchi 290", "Yasu 37", "Namikata 73a" and "Yoshida 2a" were immersed in distilled water and Delayed light emission was measured in the same manner (the integrated value of the obtained delayed light emission was DL ′ ₁₀ And ). After 4 days, 6 days, 8 days, 10 days, and 12 days after the start of the test, the delayed luminescence was measured in the same manner as described above (the integrated value of the obtained delayed luminescence was DL ′, respectively). ₁₁ , DL ' ₁₂ , DL ' _Thirteen , DL ' ₁₄ And DL ' _Fifteen And ). And DL ' ₁₁ / DL ' ₁₀ , DL ' ₁₂ / DL ' ₁₀ , DL ' _Thirteen / DL ' ₁₀ , DL ' ₁₄ / DL ' ₁₀ , And DL ' _Fifteen / DL ' ₁₀ Was determined as a comparative value. FIG. 5 shows such comparison values.
[0053]
As shown in FIG. 5, the ratio of the DL value (DL value / DL value immediately after the treatment) did not significantly differ between the test individuals (clones) over time only with the distilled water treatment. As for the samples inoculated with pine wood nematodes, the ratio of DL values (DL value / DL value immediately after inoculation) showed a large difference between individuals with the passage of days (FIG. 4). After the day, a significant difference was observed between individuals at the 1% level, and the correlation coefficient between the ratio of delayed luminescence and the degree of withering was r = -0.730.
[0054]
From these results, it was found that individuals having resistance to pine wood nematode disease and those susceptible could be distinguished by measuring the delayed luminescence of needles.
[0055]
[Relationship between degree of branch browning and delayed luminescence]
In the above “Evaluation of disease resistance (method using branches as a sample)”, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days and 7 days after inoculation, DL ₁ / DL ₀ , DL ₂ / DL ₀ , DL ₃ / DL ₀ , DL ₄ / DL ₀ , DL ₅ / DL ₀ , DL ₆ / DL ₀ , And DL ₇ / DL ₀ And the degree of browning at 1 day, 2 days, 3 days, 4 days, 5 days, 6 days and 7 days after inoculation was evaluated based on Table 4 below. Then, the relationship between the ratio of DL values (DL value / DL value immediately after inoculation) and the degree of browning was evaluated.
[Table 4]

[0056]
As a result, the correlation coefficient between the ratio of the DL value (DL value / DL value immediately after the inoculation) 7 days after the inoculation of the pine wood nematode and the degree of browning was r = -0.956 **. Therefore, it was found that the degree of browning of the branch can be evaluated by using the ratio of the DL value 7 days after the inoculation (DL value / DL value immediately after the inoculation).
[0057]
[Relationship between degree of needle browning and delayed luminescence]
In the above “Evaluation of disease resistance (method using leaves as a sample)”, 4 days, 6 days, 8 days, 10 days and 12 days after inoculation, DL ₁₁ / DL ₁₀ , DL ₁₂ / DL ₁₀ , DL _Thirteen / DL ₁₀ , DL ₁₄ / DL ₁₀ And DL _Fifteen / DL ₁₀ Was determined, and the degree of browning at 4, 6, 8, 10, and 12 days after inoculation was evaluated based on Table 4 above. Then, the relationship between the ratio of DL values (DL value / DL value immediately after inoculation) and the degree of browning was examined.
[0058]
As a result, the correlation coefficient between the ratio of DL value (DL value / DL value immediately after inoculation) 12 days after inoculation with the pine wood nematode and the degree of browning was r = -0.876 *. Therefore, it was found that the degree of browning of the needles could be evaluated by using the ratio of the DL value 12 days after the inoculation (DL value / DL value immediately after the inoculation).
[0059]
[Effect of irradiation light on the method for evaluating plant disease resistance]
The present year branches of "Tsuyazaki 50", "Misaki 90", "Kawauchi 290" and "Yasu 37" are watered in a test tube, and the degree of withering after inoculating 5000 pine wood nematodes based on the criteria in Table 1. I checked.
[0060]
On the other hand, needles collected from the current branch of "Tsuyazaki 50", "Misaki 90", "Kawauchi 290" and "Yasu 37" were cut into 3 cm lengths, and after thinly shaving the surface, 100,000 heads / 3 mL It was immersed in the pine wood nematode suspension for 20 hours. Ten needles per individual were arranged in a petri dish and left in a dark room at 24 ° C. for 6 days.
[0061]
Next, the sample was irradiated with blue light (center wavelength: 470 nm), green light (center wavelength: 550 nm) or red light (center wavelength: 650 nm) emitted from a light-emitting diode for 5 seconds using an ARGUS50 / VIM system manufactured by Hamamatsu Photonics. , The delayed light emission was measured for 0.1 second (the integrated value of the delayed light emission obtained from the blue light, the green light and the red light was DL _b6 , DL _g6 And DL _r6 And ). FIG. 6 shows the DL value of each light. The degree of withering 6 days after inoculation was determined based on the criteria shown in Table 1.
[0062]
When the correlation between the DL value and the degree of withering was determined for each of blue light, green light and red light, the correlation coefficients were r = -0.686 for red light and r = -0.610 for blue light. R = -0.084 with green light. From these results, it was found that red light and blue light were particularly effective as irradiation light when evaluating the resistance to pine wood nematodes using leaves.
[0063]
Further, the correlation between the DL value of red light / DL value of green light, the DL value of blue light / DL value of green light, and the degree of withering was determined. The DL value was r = -0.709 *, and the DL value by blue light / DL value by green light was r = -0.670 (FIG. 7). From these results, in evaluating the resistance to pine wood nematodes using leaves, red light, blue light and green light are effective as irradiation light, and the evaluation of the evaluation is performed by combining the DL values of these lights, respectively. It has been found that the accuracy is further increased.
[0064]
【The invention's effect】
As described above, according to the present invention, there is provided a method for evaluating disease resistance of a plant, which evaluates a plant having resistance (or susceptibility) to a pathogen with high accuracy in a short time without requiring heavy labor. It becomes possible to do.
[Brief description of the drawings]
FIG. 1 is a schematic diagram of a disease resistance measuring device that can be used in the present invention.
FIG. 2 is a graph showing the change over time in the ratio of delayed luminescence of Japanese pine (branches) inoculated with pine wood nematodes.
FIG. 3 is a graph showing a change over time in a ratio of delayed luminescence of Japanese black pine (branches) treated with distilled water as a control.
FIG. 4 is a graph showing the change over time in the ratio of delayed luminescence of Japanese black pine (needle) inoculated with pine wood nematodes.
FIG. 5 is a graph showing the change over time in the ratio of delayed luminescence of Japanese black pine (needle) treated with distilled water as a control.
FIG. 6 is a diagram illustrating a delayed light emission amount depending on a type of irradiation light.
FIG. 7 is a diagram showing a ratio in a case where a DL value due to irradiation with red light or blue light is divided by a DL value due to irradiation with green light.
[Explanation of symbols]
2 ... Disease resistance measurement device, 4 ... Delayed emission measurement device, 6 ... Control unit, 7 ... Output unit, 8 ... Tested plant sample, 10 ... Dark box, 11 • ..Light emitting system, 12 ... VIM camera, 16 ... light emitting unit, 30 ... sample mounting table, 32 ... lens, 33 ... filter, 34 ... light source, 36 ... -Optical fiber, 38-Shutter, 39-Shutter controller, 40-Image intensifier, 42-CCD camera, 50-VIM camera controller, 52-Image processor, 54- ..Image output monitor, 56 ... data analyzer, 58 ... data output monitor

Claims (5)

In the method for evaluating plant disease resistance to evaluate the resistance or susceptibility of a test plant to a pathogen,
A first step of inoculating the sample of the test plant with the pathogen, irradiating the sample of the test plant immediately after inoculation with light, and detecting the delayed luminescence that occurs for a predetermined time;
A second step of, after leaving the sample of the test plant inoculated with the pathogen for a predetermined period of time under predetermined conditions, irradiating the sample of the test plant with the light, and detecting a delayed emission that occurs for a predetermined period of time; ,
A third step of evaluating the resistance or susceptibility of the test plant to the pathogen by comparing the amount of the delayed luminescence measured in the first and second steps to determine a comparison value, A method for evaluating plant disease resistance, comprising: The comparison value is a value obtained by dividing the amount of the delayed light emission detected in the second step for the predetermined time by the amount of the delayed light emission detected in the first step for the predetermined time. The method for evaluating disease resistance of a plant according to claim 1. The method for evaluating plant disease resistance according to claim 1 or 2, wherein the light is white light, red light, blue light or green light. The method for evaluating disease resistance of a plant according to any one of claims 1 to 3, wherein the sample of the test plant is a branch or a leaf of the test plant. A first step of inoculating each of the samples of the two or more test plants with the pathogen, irradiating the sample of the test plant immediately after the inoculation with light, and detecting the generated delayed luminescence for a predetermined time,
After leaving the sample of the test plant inoculated with the pathogen for a predetermined period of time under predetermined conditions, irradiating each of the test plant samples with the light, and detecting the generated delayed luminescence for a predetermined period of time. Steps and
For each sample of the test plant, a value obtained by dividing the amount of the delayed light emission detected in the second step for the predetermined time by the amount of the delayed light emission detected in the first step for the predetermined time is obtained. 3 steps,
A test plant showing a value lower than the average of the values, a test plant showing a significant value by analysis of variance of the value, excluding from the two or more test plants, the resistance to the pathogen A fourth step of selecting only test plants having
A fifth step of sexually or asexually breeding the test plant selected in the fourth step to obtain a plant having resistance to a pathogen.

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