.

Appendiks 1














Macroscopic leaf symptoms

in wheat infected by Tilletia tritici








Manuscript submitted to European Journal of Plant Pathology, May 2000.














by










Anders Borgen and Lars Kristensen










Keywords: diagnosis, disease assessment, resistance, sensitivity, specificity, T.caries



Summary

Common bunt (Tilletia tritici) infect the wheat plant during germination of the seed. For experiments with common bunt it is conventional to examen symptoms of infection in the head of the plant. Previous research has advocated for the possibility to examen chlorosis on the leaf instead in order to reduce time for the duration of the experiment. Current experiment with the two diagnosis methods in climate chambers demonstrate that the quality of the two methods is depending on the wheat variety used.



Introduction

Common bunt (Tilletia tritici (Bjerk.)Wint. syn. Tilletia caries (DC.)Tul. ) is a seed borne smut disease of wheat, but it can also be transmitted through soil (Borgen 2000, in press) or agricultural equipment (Kristensen et al 1996). Instead of normal seeds in the florets, infected plants develop sori (bunt balls) containing black teleudospores.

Common bunt is a systemic disease, where infection can only occur during the germination period. The development of spores is the significant symptom for epidemiological investigations of the disease. In other types of experiments it can be of interest to find infected plants even they do not develop sori and spores in the head. This can be either because the plant escapes infection due to resistance mechanisms or because of environmental conditions.

A problem in experimental work with common bunt is that the most characteristic macro symptoms develop after heading. It is therefore only possible to make one experiment per growing season based on these late developing symptoms. An experimental method based on symptoms visible earlier in plant development would speed up the scientific process.

Churchward (1934) was the first to describe chlorotic spots on leaves caused by infection by common bunt (Tilletia tritici). Johnston and Lefebvre (1939) described the observation in more detail for wheat infected by Tilletia foetida. Kendrick and Purdy (1959), Trione (1973), Becker (1992) have confirmed the observation for Tilletia tritici. The leaf spots are already visible at the 2-3 leaf stage and are visible throughout the plant development until senescence.

In this experiment we compare an assessment of bunt infection for experimental purposes based on the earliest macrosymptoms on leaves with an assessment based on the development of bunt spores in the ovaries and discuss the implementation of the use of this method.

The concepts of sensitivity and specificity are often used to evaluate diagnostic methods in human and veterinary medicine. Here, sensitivity is defined as the proportion of true positive tests, and specificity the proportion of true negative tests (Hennekens and Buring 1987). In this experiment sensitivity can be defined as the proportion of plants with leaf symptoms which is actually infected and specificity is the proportion of plants without leaf symptom which is in fact uninfected.

For the investigation of sensitivity and specificity, the development of sori in the ovaries was chosen as the standard, since this method is very unambiguous, and it is the recommended and normally used method (Anomyme 1997).



Materials and methods

During a two years period we made 15 climate chamber experiments with common bunt (Tilletia tritici), where symptoms of infection were registered both at the stage of 4-6 leafs (Zadoks 14-16) and after heading (Zadoks 73-77). The experiments included different seed treatments, different levels of inoculum contamination and 15 different cultivars of winter wheat (Triticum aestivum) and one cultivar 'Östro' of spelta wheat (T. spelta).

Plants were seeded in 25 cm diameter pots containing 5 l of pre-fertilized peat soil (Pinstrup whole mixture 2). After seeding the soil surface was covered with a thin layer of reflective light sand to reduce the radiation-induced temperature rise in the soil. The pots were placed in a climate chamber that was programmed for 10ºC day and 5ºC night (12 h/12 h). The light intensity was 250-400 µEm-2s-1. Twelve of the chambers had pots contained 12-15 plants with 32 pots per chamber. In an additional 3 chambers each pot contained 50 plants, which after first diagnosis were transplanted into larger boxes divided according to symptoms.

The seedlings emerged after about 8-10 days, and the day temperature was decreased by one degree every week. After additional five weeks the plants were taken out of the climate chamber and diagnosed for macroscopic leaf symptoms of common bunt. The number of plants with leaf symptoms in each pot was counted, and these plants were marked. In cases of doubt the symptoms were described and the plants specially marked. The plants were hereafter placed at 5ºC for 46 days to insure vernalization and then grown to heading in a greenhouse.

After heading the number of tillers which had developed sori in the flowers were counted for each plant, and the result were compared with the first diagnosis.

Observations

In doubt cases, where the plants were marked for special observation we concluded that chlorotical spots related to infection by T.tritici are not superficial, but penetrated the leaf. Small spots can therefore often be seen easier under back lighted conditions to separate them from superficial spots caused by other means. We also concluded that plants with just a single chlorotic spot just visible to the naked eye shall be included in the group of plants with leaf symptom and are so in the presented data.

We also observed that in cases where the leaf infection level is high the leaf symptoms on the infected plants are more visible and more frequent, while in cases where the infection level was low, many plants had fewer and more weak symptoms. This observation may indicate that the number of chlorotic spots correlates with the number of infection sites. However this observation and hypothesis have not been studied further.

Chlorosis observed on the leaves differed in character between the cultivars. Some cultivars express discrete spots while others express diffuse plaques as sign of infection.



Results

The sensitivity and specificity analysis are presented in Table 1 and 2. The numbers presented in Table 1 are bulked from 16 different cultivars, the major proportion of the plants (80.2%) coming from only two cultivars. In Table 2, the sensitivity and specificity are calculated for each cultivar. This Table shows that there are differences between cultivars, and the cultivars are grouped according to their sensitivity and specificity. In cases with low sensitivity (Group C) the leaf symptom assessment will underestimate the likelihood of development of sori in the florets. In cases of low specificity (Group B) the assessment will overestimate the likelihood of development of sori and spores in the head of individual plants in these cultivars.





Table 1: Sensitivity and specificity of diagnosis of bunt infection based leaf symptoms at the 4-6 leaf stage compared with symptoms based on development of sori in the head. 2+2 table after Hennekens and Buring (1987). Data bulked from 16 cultivars.
Head infection
Leaf infection + -
+ 2,422 610 Sensitivity: 2422*100

2422+408

= 85,6%
- 408 2,119 Specificity: 2119*100

2119+610

= 77,7%




Table 2. Number of plants with and without symptoms of bunt infection when diagnosed at the Zadoks stage 14-16 on the leaves or at Zadoks stage 71-77 in the head. Sensitivity and specificity are calculated as presented in Table 1.


Cultivar
Head infection

percent


Grouping
Leaf infection + -
Kosack + 871 207 Sensitivity: 78.8



A

Sensitivity>75%

Specificity>75%

- 234 1,043 Specificity: 83.4
Longbow + 35 2 Sensitivity: 83.3
- 7 21 Specificity: 91.3
Herzog + 60 1 Sensitivity: 93.8
- 4 5 Specificity: 83.3
Husar + 1,001 230 Sensitivity: 89.0
- 124 747 Specificity: 76.5
Nova + 36 4 Sensitivity: 90.0

















B

Sensitivity>75%

Specificity<75%

- 4 9 Specificity: 69.2
Mikon + 6 26 Sensitivity: 75.0
- 2 44 Specificity: 62.9
Obelisk + 78 4 Sensitivity: 97.5
- 2 4 Specificity: 50.0
Soisson + 45 9 Sensitivity: 79.0
- 12 17 Specificity: 65.4
Salut + 30 5 Sensitivity: 93.8
- 2 5 Specificity: 50.0
Yacht + 28 46 Sensitivity: 90.3
- 3 23 Specificity: 33.3
Hereward + 21 37 Sensitivity: 100.0
- 0 12 Specificity: 24.5
Haven + 55 6 Sensitivity: 100.0
- 0 1 Specificity: 14.3
Ramiro + 65 15 Sensitivity: 97.0
- 2 2 Specificity: 11.8
Miras + 78 7 Sensitivity: 97.5
- 2 0 Specificity: 0.0
Sorbas + 12 4 Sensitivity: 57.1 C

Sensitivity<75%

Specificity>75%

- 9 122 Specificity: 96.8
Östro (spelt) + 1 7 Sensitivity: 50.0
- 1 64 Specificity: 90.1




Discussion

To evaluate the sensitivity and specificity of a diagnostic method, it is necessary to know which plants in the experiment are truly infected and which are not (Hennekens and Buring 1987). In the case of bunt infection this is very difficult. Mycelium is very difficult to find by histological investigation, so this diagnosis will not find all infected plants, and this method will destroy the plant, and thereby defeat further growth of, and experiment with the plants. Churchward found bunt mycelium in lesions caused by Ustilago tritici and wrote, that this was also the case in lesions caused by Tilletia tritici. However, this could not be confirmed by the extensive investigation by Trione (1973), who could not find the pathogen present in the plant outside the infection sites, nodes and meristematic tissue, even not in the chlorotic spots on the leaf. The course of these spots is unclear, but their presence has been shown in many varieties (Kendrick and Purdy 1959, Becker 1992) as confirmed in this experiment. Chlorotic leaf spots can be caused by other means than bunt infection e.g physical damage or infections by other pathogens. It is therefore likely that some plants will be tested falls positive, and in cases where small spots are not discovered, the test will be falls negative.

The chlorotic leaf spots at the early stage can be difficult to find, and can be confound with other chlorotic spots on the leafs. In this study some plants (14.4%) developed infected ears even we found no chlorotic spots on the leafs. Either there were no spots at the early stage or they were not observed.

The result of a diagnosis at the early stage will to some extent be influenced by the persons involved and the environmental conditions for plant growth like nutrition, water status, light etc. Even we have tried our best to limit these extrinsic factors, it can not be excluded that the differences between cultivars can be explained by the fact, that the diagnosis to some extent is a subjective judgment wether at chlorotic spot is truly a bunt symptom or caused by other means. The low sensitivity or specificity found for some cultivars may therefore be influenced by the fact that only a limited number of plants are examined in these varieties, and more experience with the expression of the symptoms for these particular varieties, would improve the sensitivity/specificity for this partly subjective diagnostic method.

The development of sori and spores is a very certain symptom for infection, even sori from the outside can be mistaken for the seed ball nematode (Anguina tritici) (Tullgren 1929). The sensitivity of the diagnosis based on head symptoms can therefore be considered as very high, likely to reach 100% in all tests.

Plants with head symptoms can be considered as truly infected, but plants without head symptoms are not necessarily uninfected. 22.3% of the plants with leaf spots did not develop sori and spores in the ears after heading. Some tillers escapes infection during later plant development, and in some plants all tillers escape infection and the plant will hence be assessed free of disease uninfected even being actually infected.

In table 1 and 2 results of the analysis of the sensitivity and specificity of the leaf symptom method are presented, where the development of sori in the head is used as standard for 'truly infected plants'. These results here must therefore be taken with the presumption that the prerequisite for the sensitivity test is true, but not completely for the specificity.

The results shows that both the sensitivity and the specificity is relatively high for most cultivars, but for some varieties it is much lower. A low specificity (i.e. many plants with leaf symptoms without symptoms in the head) can be explained by the fact that it is a combination of errors with the test method at the heading stage and the errors coursed by the leaf symptom method.

Hennekens and Buring (1987) argues that there in general is a tradeoff between sensitivity and specificity. If the sensitivity of a method is high, it will often be on the account specificity and vise versa. This does not seem to be the case in the choice between cultivars, since some cultivars express both a high sensitivity and a high specificity, while others are low in one or both.

Leaf symptoms in resistant cultivars

Bunt-related chlorosis can be observed in both resistant varieties and in susceptible varieties (Churchward 1934). Chlorotic spots in resistant varieties are also observed in case of infection by T. contraversa (Hoffmann 1982) and T.laevis (Johnston and Lefebvre 1939).

The presence of leaf symptoms in resistant varieties contradicts the findings of Kendrick and Purdy (1959) who found that resistant varieties did not show leaf symptoms at the early stage, and even recommended the use of the leaf diagnosis in screening cultivars for resistance. This contradiction may be explained by the different resistance mechanisms, since some resistance mechanisms works during infection stage, e.g. by preventing infection in the cuticula, while other mechanisms functions later in the plant development, e.g by preventing mycelium growth in the ovaria. For some of the mechanisms working late in the plant development, it is likely that plants are in fact infected in the early stage and therefore shows chlorotic symptoms on the leafs, yet the resistance prevent them from developing sori and spores in the head. We have thus seen frequent and clear symptoms in the highly resistant variety Stava with the resistance genes bt8, bt9 and bt10 (not included in this experiment), even we have never been able to see sori and spores develop in the ears of this variety. The varieties Yacht and Hereward shows many symptoms at the early stage, but a moderate infection in the head. These varieties belong to the intermediate group with some degree of undescribed resistance (Nielsen 1999). On the other hand, the variety Mikon with a moderate resistance shows equal low symptom rates at both stages. Using the chlorotic spots for screening cultivars for resistance thus have considerable disadvantages at least for some types of resistances.

Most experiments with common bunt are based on assessments based on the development of sori in the head. However, neither leaf assessment nor head assessment shows all infected plants. Diagnosis at heading stage based on the presence of spores in the sori is therefore not necessarily more certain than diagnosis at the early stage in terms of determining whether or not a plant is or have been infected by the pathogen. Wether the one diagnostic method is better than the other therefore, depend on the purpose of the experiment: If the purpose is to predict the development of spores in the head, a leaf diagnosis has uncertainties, which is depended on the cultivars used and a cultivar with a high specificity should be chosen. If the purpose of an experiment is to evaluate wether a seed treatment is preventing infection, a cultivar with a high sensitivity should be chosen, but the specificity is in this case of less importance. One may even use some highly resistant cultivars for the experiment which is not possible if the effect of a treatment is evaluated on the basis of the development of spores in the head, which does not occur in the resistant cultivars.

References

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