Macroscopic leaf symptoms in wheat infected by Tilletia tritici

 

Makroskopische Blattsymptome der Infektion von Weizen durch Tilletia tritici

 

Anders Borgen, Agro Business Park, Niels Pedersens Allé 2, DK-8830 Tjele.  E-mail: anders.borgen@scanagri.dk 

AND

Lars Kristensen, Department of Ecology, Royal Veterinary and Agricultural University, Rolighedsvej 21, DK-1871 Frederiksberg C. Denmark.  E-mail: lkr@kvl.dk

 

Summary

Common bunt (Tilletia tritici) infects the wheat plant during germination of the seed. For experiments with common bunt it is conventional to examine symptoms of infection in the head of the plant. Previous research has advocated the possibility of scoring chlorosis on the leaf instead of head symptoms in order to reduce the duration of the experiment. By comparing the two diagnosis methods in climate chambers, the current experiment demonstrated that the sensitivity and specificity of the two methods depends on the wheat variety used. Using leaf symptoms as a diagnosis for infection has considerable disadvantages when used for screening for resistance in breeding programs, but have advantages e.g. when investigating effects of seed treatments.

 

Keywords: Winter wheat; Tilletia tritici; leaf symptoms, diagnosis;

 

Zusammenfassung

Steinbrand infiziert den Weizen in der Keimung und Auflaufphase. Bei Steinbrandversuchen ist es üblich, die Symptome der Infektion in den Ähren zu untersuchen. In früheren Arbeiten wurde empfohlen, Chlorosen auf dem Blatt zu bonitieren, um die Dauer des Versuches zu reduzieren. Die hier vorgelegten Ergebnisse von vergleichenden Klimakammerversuchenzeigen dab Empfindlichkeit und Spezifität der zwei Verfahren von der verwendeten Weizensorte abhängen. Die Infektionsdiagnose anhand der Blattsymptome ist bei der Verwendug in der Resistenzzüchdung mit Nachteilen verbunden. Sie hat aber Vorteile bei z.B. Untersuchungen über die Effekte von Bekämpfungsverfahren.

 

 

Stichwörter: Winterweizen; Tilletia tritici; Steinbrand; Blatsymptome; Diagnose.

 

 

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) or agricultural equipment (KRISTENSEN and BORGEN 2001). Instead of normal seed in the florets, infected plants develop sori (bunt balls) containing black teleudospores.

Common bunt is a systemic disease, in which infection only can 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 if 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 such as temperature.

A problem in experimental work with common bunt is that the most characteristic macroscopic symptoms develop after heading. It is therefore only possible to carry out one experiment per growing season. 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 leavis ( syn. T.foetida). The observation has been confirmed for Tilletia tritici by several authors (KENDRICK and PURDY 1959, TRIONE 1973, BECKER 1992, KOCH and SPIESS 2002, JOSEFSEN and CHRISTENSEN 2002). The leaf spots are already visible at the 2-3-leaf stage and persist throughout plant development until senescence.

The concepts of sensitivity and specificity are often used to evaluate diagnostic methods in human and veterinary medicine. Sensitivity is defined as the proportion of true positive tests, and specificity as the proportion of true negative tests (HENNEKENS and BURING 1987). The current experiment compares two diagnostic methods of common bunt in wheat; assessment of leaf-symptoms and assessment of ear infection. Here, sensitivity is defined as the proportion of plants with leaf-symptoms, which later also develop infected ears. Specificity is defined as the proportion of plants without leaf symptoms, which later develop healthy ears.

 

 

 

 

 

 

Materials and methods

During a two year 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 leaves (Zadoks 14-16) and after heading (Zadoks 73-77) (ZADOKS et al. 1974). 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). Only the results from the different varieties are presented, since only these had a systematic effect on sensitivity and specificity of the diagnostic method.

 Plants were seeded in 25 cm diameter pots containing 5 l of pre-fertilized peat soil (Pinstrup whole mixture m 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 programmed for 10C° day and 5C° night (12 h/12 h). The light intensity was 250-400 µEm-2s-1.

 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. The plants were hereafter placed at 5C° for 46 days to insure vernalization and then grown to heading in a greenhouse.

 After heading the number plants, which had developed sori in one or more the ears, were counted, and the results were compared with the leaf diagnosis.

For the investigation of sensitivity and specificity, the development of sori in the ovaries was chosen as the standard, since this method is unambiguous, and it is the recommended and normally used method (ANONYMOUS 1997). The calculation of sensitivity and specificity is presented in Table 1. Confidence intervals at the 95% level in Table 2 were set up under the assumption that bunt infection was binomially distributed (exact limits, PROC FREQ, SAS ver. 8.1). Further details of experimental design are presented in BORGEN (2000b).

 

 

Results and 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 difficult to find by histological investigation (TRIONE 1973). KOLLMORGEN and BALLINGER (1987) found a good high correlation (0.91) between hyphae in leaves and development of sori in heads based on staining, but this method did not find hyphae in all infected plants. JOSEFSEN and CHRISTENSEN 2002 found that hyphae were present in leaves in a number of plants, which did not develop sori in the head and recommended to detect the presence of the fungus in the inflorescence by a nested PCR technique. However, this technique are also not able to detect all infected plants (JOSEFSEN and CHRISTENSEN 2002), and the method will destroy the inflorescence, and thereby make further growth of, and experiments with the plants impossible.

Chlorotic leaf spots can be caused by means other than bunt infection e.g. physical damage or infections by other pathogens. It is therefore likely that some plants will be tested as false positives, while in cases where the minute spots indicating infection are not discovered, the test will be false negative.

The development of sori and spores is a very certain symptom for infection, even though sori can from the outside 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.

Whereas plants with head symptoms are undoubtedly infected, plants without head symptoms are not necessarily uninfected. A large proportion (22.3%) of the plants with leaf spots did not develop sori and spores in the ears after heading. If the inflorescence is not infected by the time of elongation, the plant will not develop sori (SWINBURNE 1963) and the plant will hence be assessed to be free of disease, while actually being infected. JOSEFSEN and CHRISTENSEN (2002) confirmed that fungal mycelium was present in spotted leaves in some plant, which later did not develop sori.

In Tables 2 results of the analysis of the sensitivity and specificity of the leaf symptom method are presented for different cultivars, where the development of sori in the head is used as the decisive indicator for (epidemiologically relevant) infection. The results show that both the sensitivity and the specificity parameters are relatively high for most cultivars, but, for some varieties, one or both can be low.

In cases with low specificity (i.e. many false negative plant with head symptoms, which did not show symptoms in the leaf), the leaf symptom assessment will underestimate the likelihood of development of sori in the florets. In cases of low sensitivity (i.e. many false positive plants with leaf symptoms without exhibiting symptoms in the head), the assessment will overestimate the likelihood of development of sori and spores in the head of individual plants in these cultivars. A low specificity can be explained by the fact that it is a combination of errors with the test method at the heading stage and the errors caused by the leaf symptom method. KOCH and SPIESS (2002) investigated the development of leaf spots in nine wheat varieties. In some varieties the development of leaf spots developed later than in others, indicating that specificity may increase during plant development in some varieties. It cannot be excluded that the low specificity of some varieties found in our experiment can be explained by the fact that we assessed the symptom at a very early stage of plant development.

The current experiment is conducted under climate chamber condition. The conditions in terms of light intensity and duration, and nutrient level etc. are specially designed to make the chloritic spots easy to observe, and the frequency of other chloritic spots is small under the controlled conditions. Under field conditions the spots also develop in infected plants, but many other spots caused by wind, nutrient deficiency, insects and fungal infection etc. disturb the picture, making the method for experimental purposes unreliable.

 Bunt-related chlorosis have previously been observed in both resistant and in susceptible varieties (CHURCHWARD 1934, KOCH and SPIESS 2002). Chlorotic spots in resistant varieties have also been observed in cases 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 who even recommended the use of the leaf diagnosis in screening cultivars for bunt resistance. This contradiction may be explained by the different resistance mechanisms, since some resistance mechanisms work during the infection stage, e.g. by preventing infection through the cuticle, while other mechanisms function later in the plant development, e.g. by preventing mycelium growth in the ovaries. For some of the mechanisms working late in plant development, it is likely that plants are in fact infected at the early stage and therefore show chlorotic symptoms on the leaves, yet the resistance prevents 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) when grown from bunt-contaminated seed, even though we have never seen sori and spores develop in the ears of this variety. The symptoms do not develop when grown from seed free from spores.

Using the presence or absence of chlorotic spots in screenings of cultivars as indicator for plant resistance has thus considerable disadvantages, at least for some types of resistance.

Most experiments with common bunt are based on assessments based on the development of sori in the head (ANONYMOUS 1997). However, neither leaf assessment nor head assessment identifies all infected plants. Diagnosis at the 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 has been infected by the pathogen. Whether one diagnostic method is better than the other, therefore, depends 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 depends on the cultivars used. If leaf-diagnsis is used in this case a cultivar with a high specificity should be chosen. If the purpose of an experiment is to evaluate whether a seed treatment prevents infection, leaf-diagnosis can be more appropriate and a cultivar with a high sensitivity should be chosen, whereas the specificity in this case is of less importance. The histological and molecular techniques serve as intermediate methods. Detecting the presence of the fungus in the leaf tissue by staining (KOLLMORGEN and BALLINGER 1987) or PCR (JOSEFSEN and CHRISTENSEN 2002) will have the same disadvantages as the assessment of leaf spots, giving an imprecise prediction of development of sori in the head in some varieties. The assessment of the presence of the fungus in the inflorescence (JOSEFSEN and CHRISTENSEN 2002) will give good correlation to the development of sori in the head but will only find a fraction of the infected plants. Compared with these methods, the assessment of leaf spots is quick and hence cheap, but is unreliable under field conditions.

 

 

Cited Literature

ANONYMOUS: Guidelines for the efficacy evaluation of plant protection products. Vol 2, Fungicides & Bactericides. European and Mediterranean Plant Protection Organization. 1997.

BECKER, J.: Untersuchungen zur Bekämpfung des Weizensteinbrandes (Tilletia tritici (Bjerk.) Wint.) mit nährstoffreichen organischen Substanzen und Mikroorganismen. Dissertation. Institut für Pflanzenkrankheiten der Rheinischen-Friedrich-Wilhelms-Universität, Bonn. 132 pp, 1992.

BORGEN, A. a: Perennial survival of common bunt (Tilletia tritici) in soil under modern farming practice. Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz. 170:182-188. 2000.

BORGEN, A. b: Hvedens stinkbrand - en udfordring for principperne for økologisk plantebeskyttelse. Ph.D. thesis, KVL, Denmark. ISBN 87-988060-0-9. 136 p., 2000.

CHURCHWARD, J.G.: A note on the occurrence of seedling lesions caused by cereal smuts. Proc.Linn.Soc. New S.Wales 59:197-199, 1934.

HENNEKENS, C.H., J.E.BURING: Epidemiology in medicine. Ed: S.L.Mayrent. Boston, Mass. Little, Brown and Company cop., 1987.

HOFFMANN, J.A.: Bunt of wheat. Plant Disease 66:979-986, 1982.

JOSEFSEN, L., S.K.CHRISTENSEN: PCR as a tool for early detection and diagnosis of common bunt in wheat, caused by Tilletia tritici . Mycological Research 106:1287-1292, 2002.

JOHNSTON, C.O., C.L.LEFEBVRE: A chlorotic mottling of wheat leaves caused by infections of bunt Tilletia laevis. Phytopathology 29:456-458, 1939.

KENDRICK, E.L., L.H.PURDY: A seedling reaction of wheat indicative of bunt infection. Phytopathology 49:130-132, 1959.

KOCH, E., H. SPIESS: Characterization of leaf symptoms of common bunt (Tilletia caries) and relationship to ear attack in nine wheat cultivars. Journal of Plant Disease and Protection 109:159-165, 2002.

KOLLMORGEN, J.F, D.J. BALLINGER: Detection and morphology of hyphae of common bunt fungi (Tilletia leavis and T. tritici) in wheat seedlings. Transactions of the British Mycological Society 88:555-559, 1987.

KRISTENSEN, L., A. BORGEN: Reduction of spore spread of common bunt (Tilletia tritici) via combining equipment. Biological Agriculture and Horticulture 19:9-18, 2001.

SWINBURNE, T.R.: Infection of wheat by Tilletia caries (DC.)Tul., the causal organism of bunt. Transactions of the British Mycological Society 46:145-156, 1993.

TRIONE, E.J.: The physiology of germination of Tilletia teliospores. Phytopathology 63:643-648, 1973.

TULLGREN, A.: Kulturväxterna och djurvärden. Stockholm, 1929.

ZADOKS, J.C., T.T. CHANG, C.F. KONZAK: Decimal code for the growth stages of cereals. Weed research 14(6):415-21, 1974.


 

 

Table 1: Sensitivity and specificity of diagnosis of bunt infection based on leaf symptoms at the 4-6 leaf stage (Zadoks 14-16) compared with symptoms based on development of sori in the ear (Zadoks 73-77). 2+2 table after HENNEKENS and BURING (1987). Data, indicating number of plant in each category, is bulked from 16 cultivars (see Table 2).

 

 

Leaf

infection

 

Head infection

 

 

 

 

 

 

 

+

 

-

 

 

 

 

 

 

 

+

 

2,422

 

610

 

 Sensitivity:

 

    2422*100  

2422+408 

 

= 85,6%  

 

-

 

408

 

2,119

 

 Specificity:

 

    2119*100  

2119+610 

 

= 77,7%  

 


Table 2. Relationship between leaf and head symptoms in wheat varieties infected by Tilletia tritici. Sensitivity (proportion of plants with head symptoms which also showed leaf symptoms at the Zadoks stage 14-16) and specificity (proportion of plants without head symptoms which also had no leaf symptoms at the Zadoks stage 14-16) were calculated from data of individual plants. Values in parenthesis are 95% confidence intervals.

 

                        Sensitivity                     Specificity

Husar               89.0 (87.0-90.8)          76.5 (73.7-79.1)

Kosack                        78.8 (76.3-81.2)          83.4 (81.4-85.5)

Herzog             93.8 (84.8-98.3)          83.3 (35.9-99.6)

Longbow          83.3 (68.6-93.0)          91.3 (72.0-98.9)

Nova                90.0 (76.4-97.2)          69.2 (38.6-90.9)

Soisson            79.0 (66.1-88.6)          65.4 (44.3-82.8)

Salut                 93.8 (79.2-99.2)          50.0 (18.7-81.3)

Obelisk            97.5 (91.3-99.7)          50.0 (15.7-84.3)

Yacht               90.3 (74.3-98.0)          33.3 (22.4-45.7)

Hereward         100.0 (83.9-100.0)      24.5 (13.3-38.9)

Haven              100.0 (93.5-100.0)      14.3 (0.4-57.9)

Ramiro             97.0 (89.6-99.6)          11.8 (1.5-36.4)

Miras               97.5 (93.0-100.0)        0.0 (0.0-59.0)

Mikon             75.0 (34.9-96.8)          62.9 (50.4-74.1)

Östro              50.0 (1.3-98.7)                        90.1 (80.7-95.9)

Sorbas             57.1 (34.0-78.2)          96.8 (92.1-99.1)