Phytopathogenic Fungi and Oomycetes

Lectures 5 and 6 of OUTPACE  focused on phytopathogenic fungi and oomycetes, respectively. Kingdom Fungi is very diverse, with over 100,000 fungal species. 8,000 of these species attack plants, while a mere 300 have been linked to disease in animals. Molds and yeasts are widely distributed in the air, likely contributing to their role as the most common plant pathogen. When a fungus begins its attack on plants, the plant might change to an off-color, wilt, become susceptible to attack, decline over time if it is old, or die abruptly if young. These symptoms at first appear similar to drought or starvation. The fungal phytopathogens can be biotrophic or hemibiotrophic/necrotrophic, as we have discussed with bacteria.

Of course, the plant does not go willingly to an attack, and has a number of primary and secondary defense responses. In order to prevent initial penetration, plants rely on a cuticle, cell wall, suberin layers, and a seed coat. Furthermore, the plant has hormonal responses: utilizing Salicylic acid and Jasmonic acid pathways to fight off infection. Unfortunately for the plants, fungi have a variety of counter mechanisms. Fungi breach the physical barriers with their own physical or biochemical means, through natural openings, wounds, or by means of an insect vector. Enzymes and toxins are part of a fungi’s biochemical arsenal. Some fungi have a structure called an appressoria in order to attach and penetrate a plant surface. After penetration, a specialized complex, called haustoria, develops outside the plant plasma membrane. The haustoria are responsible for nutrient and signal exchange. The diagram below shows the progression of two different fungi on Arabidopsis.

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Lecture 6 shifts to the oomycetes classified in Chromista, a group phylogenetically unrelated to fungi, though similar in morphology and physiology. Much less diverse than fungi, there are only 500-800 species. Unlike fungi, oomycetes typically lack chitin. Instead their cell walls are made of glucans and cellulose. Spores can be sexual or asexual, called oospore or zoospore, respectively. Oospores are not motile, while the zoospore has a flagella for movement. Oomycetes are found in water and in soil, and are saprobic on both plants and animals. Some are deadly plant pathogens. Phytophtora infestans is perhaps the most famous oomycete, though you may not recognize it by that name. This species was responsible for the Irish Potato Blight, killing or displacing millions of people from Ireland. Oomycetes are not just worries of the past. Today, Phytophtora ramorum causes devastation to members of the oak family. Although it is mainly a problem in California, there is a serious fear this species will spread to the east and southern coasts, wreaking havoc on oaks. The problem has been called “Sudden Oak Death”, and could have serious consequences on a variety of ecosystems. Below is a picture showing the damage this organism can cause.

Alongside lectures, we also performed a fungal infection of Arabidopsis. Botrytis cinerea  was used as the infecting agent. The pictures below show a progression of the experiment, with some brief explanations added.

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(1) Fungal spores were mixed into a solution and later used to infect leaves. (2) shows Gita carefully selecting leaves from one strain of Arabidopsis. Two Arabidopsis strains, Col-0 and pad2,  were tested and plated separately. Col-0 is more resistant than pad2.

 

(Below) Karl separates the two strains.

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a4.pngOnce 5 leaves from each strain had been separated on a plate, we took them downstairs for infection. Infection involved using a pipette to drop 20 microliters of the previously created spore solution onto each individual leaf.

 

 

 

 

 

Below is a comparison of before and after fungal infection:

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A few days later, once we received our infected leaves back, we quantified the infection of  Botrytis cinerea on Arabidopsis. This involved classifying disease severity by the amount of yellow or grey apparent on the leaves. Amount of greying/yellowing could be quantified above, below, or between 2 and 3 mm, or by a 100% grey/yellow description, if the leaf had completely turned.  Below is a distribution of the classroom data, shown by each individuals graph displaying quantification of these disease symptoms.

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To just take one example, my graph on the bottom right shows, as expected, that Col-0 was more resistant. Overall, the Col-0 leaves were more grey than yellow (60% were completely grey). Meanwhile, all the pad2 were yellowish, with 60% having yellow greater than 3mm. Yellow color indicates greater progression of infection than grey color. This concluded our section on Fungi and oomycetes.

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