甘蔗品种黑穗病抗性评价体系的建立

        阙友雄, 许莉萍, 林剑伟, 陈天生, 陈如凯 , 李依龙

(福建农林大学 农业部甘蔗生理生态与遗传改良重点开放实验室,350002)

    为了建立甘蔗品种黑穗病抗性评价体系,选用9个引进品种,设计一个包括6个对照品种在内的田间试验。首次采用混合小种进行人工浸渍接种,通过整个新植蔗生长季病害进展曲线下的面积、茎感染率和株感染率3个病情指数,以及病害流行学参数潜伏侵染期和持续发病期的分析,对其抗病性进行评价,在分析以上参数相关程度的基础上,引进了系统聚类分析法进行进一步评价。结果显示:9个引进甘蔗品种中,ROC26属于感病品种,其余属于抗病或高抗品种;3个病情指数和持续发病期的两两相关均为显著正相关,潜伏侵染期与这些参数的相关为负相关,但未达到显著水平;在类间距离大于1.0的条件下,可将15个品种聚为6类,进一步明确了各品种抗黑穗病性的相似程度;抗性鉴定标准对照种NCo310、F134、NCo376和Ya71-374的应用,明确了接种源为小种1和小种2,通过一次接种试验,明确了供试品种对2个小种的抗性水平,标准对照种的抗性表现,还说明了本生长季发病条件基本是适宜的。试验结果表明,本文建立的甘蔗品种抗黑穗病评价体系是切实可行的。

关键词:甘蔗品种  黑穗病  抗性评价  相关系数 聚类分析

Establishment of Evaluation System of Smut Resistance for Sugarcane Varieties

QUE You-xiongXU Li-pingLIN Jian-wei,CHEN Tian-sheng CHEN Ru-kai,LI Yi-long

(Key Lab of Sugarcane Eco-Physiology & Genetic Improvement, Ministry of Agriculture, Fujian Agriculture and Forestry University ,Fuzhou 350002)

Abstract  In order to establish the evaluation system of smut resistance for sugarcane varieties, nine introduced sugarcane varieties were chosen and a field experiment including six other control varieties was conducted. By means of manual dipping inoculation, smut resistance for tested sugarcane varieties was evaluated on the basis of analyses on three infected indexes of the area under disease progress curve, infected rate of stalk, infected rate of plant and on two disease epidemic parameters of latent infection period (LIP) and sustained disease duration (SDD). Systemic cluster analyses based on correlation coefficients of these parameters showed that ROC26 was sensitive to smut infection and the other varieties were resistant in total of nine introduced varieties. Positive significant correlation between three infection indexes and SDD, and negative insignificant correlation between infection indexes and LIP were found. Fifteen sugarcane varieties could be clustered into 6 clusters on the basis of cluster distance being more than 1.0. It resulted in more clear understanding of smut resistance in tested sugarcane varieties. It suggested that smut race 1 and race 2 were included in inocula and the condition for Ustilago scitaminea infection was suitable in the crop season by setting up the standard varieties of NCo310, F134, NCo376 and Ya71-374 for smut identification.

 

In addition, the results of sugarcane varieties resistant to smut race 1 and race 2 were known by only a simple field trial. Form above, a feasible evaluation system for sugarcane smut resistance was established.

Key words: Sugarcane varieties Smut (Ustilago scitaminea Syd.)  Evaluation of resistance  Correlation coefficient  Cluster analysis

 Sugarcane smut incited by fungus of Ustilago scitaminea is a worldwide disease [1]. It infects sugarcane through the bud, then the mycelia permeate the bud scale and twist the meristem zone until the elicitation of the smut whip which contains thousands of chlamydospore. The chlamydospore then spread into other buds and caused serious losses in yield and in sucrose content. As one of the main disease in mainland China, several methods are available to control smut disease, but resistance cultivars are the most effective [2]. The accuracy and efficiency of resistance identification directly influence the process of resistance breeding [3]. Although there are several methods being used in the evaluation of smut resistance of sugarcane, such as structural characters of buds assay[45], tissue invertase assaychemical assay[6], histological staining, and ultra-structural assay, only resistance identification by artificial dip-inoculation and inspection of tested material planted in field in full growth season is popular in the world[3]. Recently, marker-assisted selection (MAS) provides a new approach to selection of crop target characters. But application of this technology should be based on the establishment of technology platform containing series of linkage markers, which we still haven’t established. So, up to now, the measure of resistance identification of sugarcane varieties is still the artificial inoculation and plant breeding [7].

In this study, the resistance identification of the varieties introduced in National 948 Project were performed so as to construct the evaluation system of smut resistance for sugarcane varieties and at the same time further provide the information about disease resistance of these tested varieties.

 

1  MATERIALS AND METHODS

1.1 Tested materials

1.1.1 Sugarcane varieties

Total of 15 sugarcaneSaccharum sppvarieties, which contains five introduced in National 948 Project( HoCP91-555HoCP93-746HoCP93-750CP85-1308CP89-1509RF93-244 RB72-454ROC25 and ROC26),four standard control varieties NCO376, NCO310, F173 and F134 ,the national uniform control variety ROC10 and the local control variety Mingtang70-611 were used as tested plant materials.

1.1.2 Tested inocula

 Teliospores of Ustilago scitaminea race 2 and race 1 both were collected from Guangxi and Fujian, respectively, mingled together. They were air-dried, sealed in plastic bags and stored at 4℃. Prior to use, germination of the spores was checked [1, 3].

1.2 METHODS

1.2.1 Inoculation and field design

     Artificial inoculation and field breeding were conducted to identify the resistance [3]. Teliospore suspension contained 5×106 spores/ml[3] were prepared, which was about 40 spores in the 60× microscope, note that in the preparation process using some washing soda as surface activator which could ruin the surface tension and prevent the spores from floating on the surface. All the sugarcane stalks were cut into two-bud sets and dip-inoculated by immersion in the prepared teliospore suspension for ten minutes. Then the inoculated sets were incubated at 25℃ under humid condition for 24 hours before being planted in field. Each one-row plot was 3.5 m long with 1.2m space between plots within rows. Plots were arranged in a randomized complete-block design with two replications (plots). Thirty sets for every kind of material were inoculated. There were twenty buds in every plot.

1.2.2 Data collection and statistical analysis

After the neat emergence of seedlings, check the emergence number of each plot and begin the investigation of the infection situation when the first whip-like sort appears. Data collections were made every 15 days until whip-like sorts did not produce. The appearance date of the first whip, the number of infected stalks (including the flank tiller bud), the number of total stalks (including the flank tiller bud) and the number of infected plants (clump) were recorded. In order to judge new whip, every whip was labeled with cards on which the appearance date and the sequence number of the plant and stalk were recorded. The final cumulative infected stalks (Ymax) were used for the determination of the resistance reaction type. After the standardization conversion of the data in every index , systematical clustering of the tested materials was performed by using UPGMA (unweighted pair group method using arithmetic average).The five indexes used in cluster were LIP(latent infection period),SDD(sustained disease duration), IP(the cumulative percentage of infected plants),IS(the cumulative percentage of infected stalks) and AUDPC(area under disease progress curves) [3].The formula of AUDPC is as follows: AUDPC = (IS1 + IS2)/2 ×(t2-t1), where IS1 and IS2 is the adjacent investigated IS, respectively, while the t2 and t1 are the adjacent investigation time. Both correlation and cluster analyses were completed by Systemic Analysis Soft (SAS) 3.0. Before cluster, standardization conversion of all data used was conducted. The formula of range standardization value is as follows [3]:


 where Xij is the observation value of parameter j of clone i, Xjmin is the minimal value of parameter j of all clones, Xjmax is the maximal value of parameter j of all clones. For those parameters which exhibited negative correlation with the resistance character, the standardized data were further converted by using the formula of  X’ij=1-Xij .

1.2.3 Ratings of smut resistance

According to the rating standard put forward by predecessor, the rating of smut resistance is showed in Table 1.

Table 1 Smut resistant ratings in sugarcane

Total rate of infected stalk in plant cane

Total rate of infected stalk in ratoon

Resistance rating

Resistance

reaction type

0-3

0-6

1

HR

4-6

7-12

2

R

7-9

13-16

3

R

10-12

17-20

4

MR

13-25

21-30

5

MS

26-35

31-40

6

S

36-50

41-60

7

S

51-75

61-80

8

HS

76-100

81-100

9

HS

2 RESULTS AND DISCUSSION

2.1 Correlation analysis of infection index

Results of correlation analysis among IP, IS and AUDPC were given in Table 2. It showed that there existed significant positive correlations at 0.01 levels between every two of infection indexes of IP, IS and AUDPC, and the same level of significant positive correlations between every infection index and disease epidemic parameter of SDD was also found. And the correlation coefficients between every two of AUDPC, IS, IP and SDD were all positive. Although there existed negative correlations between LIP and the infection index IS, IP and AUDPC. The correlation coefficients were somewhat large, but they did not reach the significant level of 0.05, so was the correlation coefficient between LIP and disease epidemic parameters SDD.

Table 2 Correlation coefficients between disease epidemic parameters and infection indexes

Parameters

LIP

SDD

IP

IS

SDD

IP

IS

AUDPC

-0.666

-0.617

-0.646

-0.635

 

0.929**

0.937**

0.921**

 

 

0.943**

0.918**

 

 

 

0.987**

Notes: IP is the percentage of plant infected, IS is the percentage of stalk infected, AUDPC is the area under the disease progress curves, LIP is latent infection period, SDD sustained disease duration. Significance at 0.05 and 0.01 levels is marked with * and **, respectively. (r0.05=0.763r0.01=0.840)

2.2   The resistance reaction types of tested materials

All of the 15 sugarcane varieties tested were infected and seemed to have different levels of infection. The corresponding results of infection indexes and disease epidemic parameter were showed in Table 3.

As for infection indexes, the IP of Ya71-374 is maximal, while the maximal value of IS and AUDPC existed in ROC26, so Ya71-374 and ROC26 were the two most susceptible. According to the final cumulative IS, the resistance reaction types of the tested 15 varieties were as follows: 5 high resistance, 4 resistance, 3 susceptibility and the surplus 3 are middle resistance .There was no any tested variety exhibiting high susceptibility. In the identification system of smut resistance, the resistance reaction type of F134 and NCo310 was middlingly susceptible and susceptible, respectively. The result accounted for that there existed both Ustilago scitaminea race 1 and race 2 in the tested inocula.  Considering the conditions for

Table 3  Performance of smut resistance of tested 15 varieties

Varieties  

LIP/d

SDD/d

IP/%

IS/%

AUDPC

Resistance reaction type

ROC25

93

31

7.1

3.2

2.89

HR

70-611

63

60

15.7

12.4

6.29

MR

RB72-454

48

23

7.3

5.4

3.62

R

CP89-1509

63

54

13.7

7.1

3.08

R

HoCP93-746

30

1

5.6

2.9

0.30

HR

ROC26

18

120

42.6

41.9

25.06

S

NCo376

108

1

5.9

1.8

0.27

HR

Ya71-374

18

120

52.5

35.5

15.20

S

HoCP93-750

131

1

1.5

1.0

0.30

HR

CP85-1308

15

1

1.6

1.1

0.16

HR

RF93-244

93

1

13.3

7.0

1.05

R

F134

18

120

44.7

23.8

23.13

MS

HoCP91-555

108

16

9.5

6.7

1.45

R

ROC10

18

105

22.2

25.0

10.50

MS

NCo310

18

120

43.3

33.3

15.00

S

Ustilago scitaminea infection and the development of smut, the highly susceptible standard control variety Ya71-374 only appeared to be middlingly susceptible, and the susceptible standard variety F134 also only appeared to be middlingly susceptible to race 2, the condition appeared not to be adequate according to the resistance reaction type of the two tested varieties, so the identification resistance reaction type should have been overestimated. On the other hand, the resistance reaction type of susceptible standard control variety NCo310 was accorded with expectation, so the condition for infection and smut development was adequate accordingly and the identification result was suitable. According to the above consideration, the smut infection and development condition of this growing season was basically adequate.

2.3 Cluster identification of smut resistance of tested materials

When using LIPSDDIP and IS as cluster indexes, the tree map for UPGMA of 15 tested sugarcane varieties was produced and showed in Figure 1.At the distance of 1.0, the 15 tested varieties were clustered into six groups. According to the cluster result, cluster 1 included 3 varieties,Ya71-374, NCo310and ROC26. Cluster II included only one variety134 and cluster III included the single variety ROC10.Cluster IV included 2 varieties Mingtan70-611 and CP891509. Cluster V included 3 varieties, CP85-1308, HoCP93-746 and RB72-454, and cluster VI included 5 varieties, NCo376, HoCP91-555, RF93-244, ROC25 and HoCP93-750. The resistance of cluster I and cluster VI were the weakest and strongest, respectively. The resistance character was gradually increased from cluster I to cluster VI.

                                                                                          

                                                                                             

                                                                                          

                                                                                          

                                                                                           

                                                                                           

                                                                                         

                                                                                          

                                                                                        

                                                                                            

                                                                                          

                                                                                             

                                                                                             

                                                                                              

                                                                                       

                                                                                                

                                                                                         

 

 

By using LIPSDDIP and AUDPC as cluster indexes, the cluster result of the 15 tested sugarcane varieties was showed in Figure 2. Set the same classification standard above, the 15 tested varieties were also clustered into six groups. And the result is as follows: cluster 1 included 2 varieties,Ya71-374 and NCo310. Cluster II included 2 varieties ROC26 and F134.Cluster III included only one variety ROC10.Cluster IV included 3 varieties Mingtan70-611 , CP891509 and RB72-454. Cluster V included 2 varieties, CP85-1308 and HoCP93-746. Cluster VI included 5 varieties, NCo376, HoCP91-555, RF93-244, ROC25 and HoCP93-750. The resistance of cluster I and cluster VI were the weakest and strongest, respectively. From cluster I to cluster VI, the resistance character was also gradually increased.

                                                                                             

                                                                                              

                                                                                               

                                                                                               

                                                                                                                       

                                                                                                          

                                                                                            

                                                                                                               

                                                                                            

                                                                                                 

                                                                                                    

                                                                                               

                                                                                           

                                                                                                 

                                                                                                

                                                                                                          

                                                                                                             

                                                                                              

2  15个品种的聚类树状图

3 DISCUSSION

Generally, the identification of resistance reaction types of sugarcane to Ustilago scitaminea smut is based on the final cumulative IS all through the growing season [3]. For two reasons, setting the standard control varieties is indispensable to the artificial inoculation and field breeding. Firstly, because the resistant expression maybe influence by external condition, the identification results of standard control varieties are helpful to determine whether the condition for smut infection and development is adequate or not. Secondly, the existence of standard control varieties is good for the verdict of Ustilago scitaminea smut race. Nowadays, in mainland China,there exist race 1 and race 2 in the sugarcane planting area at the same time, so application of only one smut race can’t assess completely the resistance of sugarcane genotypes to the both races. For the first time, the commingling races were used for inocula in this study to evaluate the resistance of tested materials to both races at one time, and the identification efficiency was improved.

The introduction target of cluster analysis is to better understand the similarity of relevant resistance parameters and to demonstrate the resistance dissimilarity of various varieties. The aim has been achieved by clustering with four parameters LIP,SDD,IP and IS, which produced basically similar results for using other four parameters LIPSDDIP and AUDPC.  AUDPC came from the increasing value of IS, and was a dynamic parameter that involved the whole process of disease infection and development. Moreover, AUDPC can reflect the total disease epidemic volume of the whole growing season. Despite of that, the substitution of AUDPC for IS in cluster reduced the homologous results.

From above, it was known that the condition for smut infection and development was adequate in the present study, so the research results objectively reflected the resistance levels of various genotypes. Due to this pointROC26 was sensitive to smut infection and the other varieties were resistant in total of nine introduced varieties. ROC26 was introduced through folk channel and recommended by predominant sugarcane breeding regions because of its new code. Through the execution of National 948 project, ROC26 was exchanged and resistance was identified. The resistance identification result of ROC26 is far from optimistic, so high attention should be paid for its further use in production.

   In a word, with the establishment of the evaluation system of smut resistance for sugarcane varieties, resistance identification for smut will be more convenient and economical.

 

References

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2.       Xu L P(许莉萍),Chen R K(陈如凯). Current status and prospects of smut and smut resistance breeding in sugarcane.Fujian J.of Agri.Sci.(福建农业学报), 2000, 15 (2): 2631

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5.       Padmanaban P, Alexander K C, Shanmugam N. Studies on certain characters associated with smut resistance. Indian Phytopath. 1988, 41:594598.

6.       Lloyd H L, Naidoo G. Chemical assay potentially suitable for determination of smut resistance of sugarcane cultivars. Plant Dis., 1983, 67:11031105

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