Screening for sources of resistance

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Whiteflies and their eggs

For further reading, description of the screening for resistance.

Resistance to Bemisia tabaci in tomato wild relatives Firdaus et al. 2012. Euphytica 187:31–45

Introduction

Tomato is threatened by many diseases (fungal, bacterial, viruses and pests) and in many cases no resistance is present in the cultivated tomato genepool in Europe and therefore new sources of resistance have to be found in its wild, crossable relatives. Sources of resistance were successfully looked for in the centre of origin of the tomato in South America and many of these resistances were successfully introduced in modern varieties.

Develop a reliable screening method for whitefly resistance

Two types of assessments can be used to evaluate whitefly resistance in tomato plants: a free-choice test or a no-choice test. In a free-choice test, whiteflies are given the choice between two or more different plants, whereby the whitefly is able to choose the most preferred one.  

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Clip-on cage test

To test the resistance of our tomato plants in this experiment we will use the clip-on cage test, a no-choice test.

Plants are infested with whiteflies six weeks after sowing. Five synchronized whitefly females (one to two days old) (n) were anesthetized with CO₂ and put into a clip-on cage (2 cm in diameter and 1 cm in height) and placed immediately on the abaxial of a leaflet of the 3^rd or 4^th leaf from the apex. Five clip-on cages with each 5 females were attached per plant. Four days after infestation (d) the clip-on cages were removed from the leaves and the dead and living whiteflies (m) were counted. The number of eggs (e) was counted under a stereo microscope (10x magnifications). The clip-on cages were reassembled at their original positions on each leaflet before new adult whiteflies started to emerge from the eggs. From these tests the Adult Survival (AS) and the Oviposition Rate (OR) of the white flies are calculated. These parameters are used to determine the level of resistance of a plant.

n:  is total number of synchronized whitefly females (one to two days old) Five clip-on cages with each 5 females were attached per plant.

d:            number of days after infestation

m:           total number of living whiteflies 

e:            total number of eggs

AS:         Adult Survival

OR:         Oviposition Rate

Difference between male and female whiteflies. Click to enlarge. 

It is very important to determine a reliable score of the level of resistance of each plant. The better and more precise the phenotypic data, the better the linkage analysis can be performed.

Questions phenotyping

3.1   Antibiosis and antixenosis are two methods to combat whiteflies (look for the definition in Google or other search engines). What is the difference between the two methods?

3.2    What is the difference between free-choice and no-choice for antibiosis and antixenosis

3.3   The whiteflies are synchronized, what is the reason for this. How would you do that (be creative). The whiteflies are growing in big cages!

3.4   Male and female whiteflies are separated. For which parameter (adult survival or oviposition rate) is this needed and for which one not? Is the separation difficult?

3.5   A while after egg deposition both white and black eggs can be seen. What is the difference? What parameter can be deduced from the ratio white vs black eggs.

3.6   Whiteflies lay the eggs sometimes in circles (see picture). Any idea what is the reason for that?

3.7   In the free choice test Amaranthus plants are present. They are very susceptible for whiteflies. What is their role in the experimental set-up. Think of a method to make the whiteflies leave the Amaranthus plants.

3.8   The formulas for adult survival (AS) and oviposition rate (OR) are

Experiment: Ten synchronized female whiteflies, are in each clip-on cage. After three days the living and dead whiteflies are counted. The number of eggs is counted on the leaf area under the clip-on cage.

On plant 1:  Five whiteflies have died after three days and in total 40 eggs have been laid. Calculate OR and AS.

On plant 2: One whitefly has died after three days and in total 110 eggs have been laid. Calculate OR and AS.

Which plant is more resistant to whiteflies?

Search for the best donorplant with wildfly resistance

In the article of Firdaus et al. 2012, several tomato accessions* were tested with the clip-on cage test, see results in table 1. *An accession is a collection at a certain location of a certain species. It is a collection of different seeds. Not all plants of an accession are necessarily genetically identical.

Questions donorplant

3.1   Which accession has the highest resistance level against white flies? Explain your answer.

3.10  Which 3 accessions of different species have the highest resistance level?

The role of trichomes in resistance (Click for better quality) 

Glandular trichomes in section Lycopersicon. Wild accessions have high densities of glandular trichomes that confer resistance to several pests. Panel (A) shows the leaflet surface of Solanum habrochaites acc. LA 1777 with high densities of glandular trichome types IV and VI (B), and type I (C). Surface of Solanum pennellii acc. LA 716 is also covered by type IV trichomes (D, E) producing and secreting acyl sugars. This accession also has type VI trichomes, but in low density (F). Panel (G) shows the surface of Solanum lycopersicum cv. Moneymaker. Cultivated tomato has low density of type VI trichomes (H) and type I trichomes. Sometimes, type IV-like trichomes (I) are observed on stems, veins, and on the leaflet edges. White bars represent 500 μm in panel A, C, D, and G. In panels B, E, F, H, and I, bars represent 50 μm.

Firdaus et al. 2012 table 2 show the means of whitefly resistance parameters and type-trichome density in clip-on cage tests with several accessions of different tomato species.

Questions trichomes

3.11   What are trichomes?

3.12   Why does a plant have trichomes?

3.13  What is the difference between a type IV and a type V trichome?

3.14   What type of trichome do you think might play a role in resistance? Why?

3.15   Is there a relation between the level of resistance of an accession and the presence of

type IV or type V trichomes according to the results in Table 2?

3.16   What 3 species/accessions are the most resistant to whiteflies in Table 2? Are they the same as Table 1?

Crossability with tomato

To know the phylogenetic relationship between the resistant accession and cultivated tomato (S. lycopersicum) is very important. The more related these species are, the easier they are to cross for making large mapping populations. In the paper of Marcela Viquez-Zamora (BMC Genomics201314:354) the phylogenetic relations between different wild relatives of tomato have been calculated. See figure below.

Questions phylogeny

3.17  What 3 species are most closely related to cultivated tomato?

3.18  What 3 species are most distantly related to cultivated tomato?

3.19  Now that you know the relationships between the Solanum species and if you consider the three most resistant species/accessions from Table 1 and 2, what resistant species/accession would you choose for crossing with tomato to make a segregating population for further research?