Using a Plant Pathogenic Fungus to Demonstrate Koch's Postulates

I: How-To-DolIta

Fun Microbiology:Using a Plant Pathogenic Fungus to DemonstrateKoch's Postulates

James K. Mitchell Kathy M. Orsted Carl E. Warnes

Robert Koch (1843-1910) was a Ger- periment we will describe a method of 4. Prepare serial dilutions. man physician who made many contri- teaching Koch's postulates by using a 5. Grow and inoculate plants. Downloaded from http://online.ucpress.edu/abt/article-pdf/59/9/574/48133/4450385.pdf by guest on 29 September 2021 butions to medical microbiology, in- plant pathogenic fungus that infects a 6. Isolate a pure culture using agar cluding his important discoveries of specific weed. streak plates. the bacteria responsible for causing an- The fungus Colletotrichumgloeospori- 7. Demonstrate the four steps of thrax and tuberculosis (Prescott et al. oides (Penz.) Sacc. f.sp. aeschynomeneis Koch's postulates. 1996). During his investigations of a pathogen of northern jointvetch, Ae- these two diseases, Koch was credited schynomenevirginica (L.) BSP., a legumi- with establishing a series of criteria for nous weed (Daniel et al. 1973). Use of Materials & Methods proving the causal relationship be- this microbe (ATCC 20358) as a bioher- tween a pathogenic microbe and a spe- bicide was patented in 1974 (US Patent Inoculum Production cific disease. These steps, known as No. 3,849,104) and subsequently mar- Stock cultures of the fungus are Koch's postulates, are the following: keted as the product COLLEGOTM maintained on potato-dextrose agar Bioher- 1. The microorganism should be (Upjohn Co., Kalamazoo, MI). (PDA) slants under mineral oil at 7? C bicides are commercial formulations of present in all hosts suffering from or glycerol-skim milk at -80? C. PDA viable fungus (mycoherbicide) spores the disease and absent from all slants should be subcultured every 4 - 6 or bacteria (bactoherbicide) that kill healthy hosts. months. If this experiment is con- specific weeds as effectively as chemi- 2. The suspected microbe must be ducted once per year, a new culture isolated from the diseased host cal herbicides (Anderson & Walker should be purchased (American Type 1985; Joye 1990; Kirkpatrick et al. 1982; and grown in pure culture. Culture Collection, Rockville, MD) for 3. The same disease must result Mitchell 1988; Mitchell 1993a & 1993b; each experiment or a streak plate from when the isolated microbe is inoc- Riddle et al. 1991). The host is killed by previous Koch's postulate experiment ulated into a healthy host. means other than innate toxicity, but should be used to prepare additional 4. The same microbe must be reiso- by interaction of the host-pathogen me- PDA slant stock cultures. Conidia lated from the experimentally in- tabolism (Charudattan & Walker 1982). (spores) of C. gloeosporioidesfor inocu- fected host and shown to be iden- Consequently there is no toxic residue lating plants are produced within a tical to the original isolate. in the crop, buildup in the soil, water sucrose-V8 broth medium: or food chains or other adverse effects These four postulates, which are ap- on the environment as observed with * 10 g KNO3 plicable to most plant, insect and ani- chemical herbicides (Lorenzi & Jeffrey . 5 g KH2PO4 the basic mal diseases, still form 1987). * 2.5 g MgSO4 x 7H20 method for determining that a particu- Each weed (plant) species is known * 20 g sucrose is caused by a given micro- lar disease to be a host of several highly specific * 0.02 g (trace) FeCl2 organism. As educators, many of us fungal and bacterial pathogens. These * 150 ml V8 juice it difficult to demonstrate Koch's find types of plant pathogens will infect a * 850 ml tap water. postulates and principles of disease specific host plant species and not control when working with animals. cause any harm to other plants or bio- This medium is adjusted to pH 6.5 Not only is feeding and upkeep a time- logicals, including humans and other with 50% NaOH (w/v) and 50 ml ali- consuming process, but dealing with mammals. This specificity suggests quots placed into 250-ml Erlenmeyer moral issues involving experimenta- their use as a safe and easy means by flasks fitted with cotton plugs. Steril- tion with live animals often hinders which to demonstrate Koch's postu- ized flasks are inoculated with a loop- student creativity. In the following ex- lates within the classroom. ful of the fungus and placed on a rotary Students conducting this experiment shaker adjusted to 155-200 rpm at will learn to: room temperature (26-280 C). Follow- James K. Mitchell is an Associate Pro- ing sporulation, conidia within broth fessor, Kathy M. Orsted is an Under- 1. Follow aseptic techniques. are from graduate Student, and Carl E. War- separated fungal mycelium nes is a Professor in the Department 2. Grow and produce spores of a by filtering the culture through four of Biology, Ball State University,Mun- fungus. layers of cheesecloth or cotton gauze. cie, IN 47306-0440. 3. Use a hemacytometer for enumer- All glassware should be free of soap ating spores. and rinsed thoroughly with either tap,

574 THEAMERICAN BIOLOGY TEACHER, VOLUME 59, NO. 9, NOVEMBER/DECEMBER1997 tant (detergent) Tween 20 is nontoxic to most fungal spores and plants (at above concentrations)and is used to lower surface tension in order that the inoculum will not be repelled by the waxy cuticle of the leaf. Commercial detergents (e.g. hand, clothing and dishwashing soaps) contain ingredi- ents that are toxic to fungal spores and should not be used when preparing dilutions. Two methods for preparing a specific spore titer are herein described. Assume that the originalspore suspen- - - I- -. - - ,------I - - sion (broth filtrate)has a titer of 3.4 x 107 spores/ml and we wish to prepare an inoculum with a titer of approximately 1 x 106 spores/ml: "Method A" Downloaded from http://online.ucpress.edu/abt/article-pdf/59/9/574/48133/4450385.pdf by guest on 29 September 2021 titer of higher concentration titer desired of lower concentration = dilution factor 3.4 X 107 1. =34 1X106 2. 1 ml of original (higher) + 33 ml diluent (Tween solution) = 34 ml total volume. To check if math is correct;(1/34) x (3.4 x 107) = 1 x 106 spores/ml. 0.2 mm 3. To prepare 75 ml of inoculum with titer of approximately 1 x 10' spores/ml: 1.0 mm 1 X - = - cross multiply and solve X Figure 1. Hemacytometergrid system. 34X = 75 75 deionized or distilled water. Through- centration) is composed of the 16- X = = 2.21 ml original out these procedures, aseptic tech- smallest squares found within the grid niques (e.g. flaming inoculating loops system (Figure 1A). There are 25 such + 72.79 ml diluent and pressure cooking or autoclaving sized squares in Figure 1. Spores from B" culture glassware and media at 15 psi 5 of these squares (Figure 1A-E) are "Method for 15 minutes) should always be fol- usually counted at 400-450X magnifi- (X/ (X + Y)) x (T1) = T2 lowed to prevent the contaminationof cation, which will provide sufficient cultures, sterile media, and/or infec- replicationwhen determiningtiter of a re-arrangingequation to T1X= T2(X+ tion of plants. sample. Y) or To determine spore titer of an un- TjX = T2X + T2Y Using a Hemacytometer known sample, count total # spores T1 = original titer of higher concen- The hemacytometerwas developed found in A + B + C + D + E and tration mathematical to count red blood cells under the multiply this sum by the T2= titer desired of lower concen- X in microscope, but may also be used to factor 5 104. This will result units tration the accepted count the spores or yeast cells of most of # spores/ml of sample, X = volume of original titer a microbe con- fungi. It is a glass slide composed of a method of describing Y = volume of diluent (e.g. Tween 20 grid system with different sized centrationin liquids. solution) x W) squares of known dimensions (L Preparing Dilutions 1. Whatvolume of original(X) must be etched upon the surface (Figure 1). A added to 75 ml diluent (Y) so that a coverslip is placed on the slide to pro- The filtrate from sporulated broth final titer of approximately1 X 106 vide a depth (height) of 0.1 mm so that cultures is diluted in nonchlorinated spores/ml results? the volume within a certain size cube (e.g. distilled or deionized) water 2. (3.4 x 107) (X) = (1 X 106) (X) + (1 X (L x W x H) may be determinedwhen amended with Tween 20 (0.05-0.1 %, 106) (75) a liquid sample is added to loading v/v) which is pre-cooled to 7?C after 3. 3.4 x 107X = 1 X 106X + 7.5 x i07 groove. mixing. Using chilled diluent prevents 4. 3.3 x 107X= 7.5 x i07 The size square most often used for spores from germinating before they 7.5 x i07 determining fungus spore titer (con- are inoculated onto plants. The surfac- 5. X = 3 0 = 2.27 ml of original

FUN MICROBIOLOGY575 To prepare more dilute spore titers (e.p. 1 x 103 to 1 x 10 spores/ml), one could use the 1 x 106 sample as the next .~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... !.i'il''!':::;....iiw}'.u ...... stock solution from , . | ! .'.''.':: t.4 '}: . i ' X ', ' '" ' .. : ? : :~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... ' ..... : 'F'~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.....'::::'.....":':.:..-.:.,b_ I .. which to prepare 1:10 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... Pe.:...... s.: ::.:.....: ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... ,',:w.^...... vy * serial dilutions. For _~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... ,UE,j...... :..._a 4 _w. example, pipet 10 ml from 1 x 106 sample . . ' .4 ...... , , . into 90 ml diluent and mix well to pre- ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... ''.:::S,,...... ,.,,,.,.....P' ' ! ' { .. pare 100 ml of 1 x ...... ~~~~~~~~...... * s _.'... ' 105 spores/ml solu- :...... ::'.:r.W^.'...... s''^'...... s...... ,.. ' _ tion (see "Method ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... :..'..,.'..'.''..,,'''''''.;'_ _.... B"). Next, pipet 10 ml from this latter sam- l...... ;.bz~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~;Z...... xND -s ...... ple into 90 ml diluent ...... ;;S _ ...... and continue with Downloaded from http://online.ucpress.edu/abt/article-pdf/59/9/574/48133/4450385.pdf by guest on 29 September 2021 1:10 serial dilutions until you prepare a range of spore titers you are interested 2. Koch's (a) Lesions of on northern stems. Arrow indicates in mix- Figure postulates. fungus jointvetch spore testing. When mass. Bar = 1 (b) view of at 430X magnification.Bar = 30 ,um. ing dilutions, swirl cm; Microscopic fungal spores or vortex gently to avoid excessive foam- ing. It is advisable to use fresh pipets 100% RH), inoculated seedlings are en- A flame-sterilized dissecting probe is and rinsed glassware for each titer to closed within clear plastic bags con- used to remove a portion from a avoid spore carryover. taining moistened paper towels and single spore mass when viewing un- placed on the greenhouse bench or der a stereomicroscope. This is asep- Plant Production & Inoculation placed into a dew-deposition chamber tically transferred to the surface of a adjusted to 26?C. After 24 hours, plants PDA plate and streaked for isolation Seeds of northern jointvetch (also are removed from their respective hu- with an inoculating loop. To improve known as curly indigo) may be col- midity chamber, placed upon the the successful isolation of this fun- lected from plants growing within the greenhouse bench and observed daily gus, the agar medium should be field or possibly from weed special- for symptom development. amended with streptomycin sulfate ist(s) located at the state land-grant (100 ,ug/ml) after sterilization and/or college in your area. Seeds may also be Koch's Postulates adjusted to pH 6.0 prior to sterilization. purchased from Valley Seed Service This added precaution will lessen the (Fresno, CA). Experimental plants are Leaf and/or stem tissue exhibiting chance of bacterial contamination. grown on a greenhouse bench for 4-6 disease symptoms are placed into petri Characteristic cottony light-orange col- weeks (10-15 cm height) at 26-30? C in dishes containing moistened paper onies of C. gloeosporioides should ap- 7.5 cm diameter plastic pots (one towels (humidity plates) to promote pear within 3-5 days when incubated plant/pot) containing one part field sporulation of the fungus. Each speci- under the same environmental condi- soil and three parts commercial potting men should be excised such that 1 cm tions as humidity plates. mix. Control (surfactant only) plants of healthy tissue remains adjacent to To complete Koch's postulates, a are sprayed first, followed with lowest diseased tissue. Growth and sporula- portion from a single isolated colony is to highest spore titers. By proceeding tion of this fungus often appears within aseptically removed from an agar from lower to higher spore concentra- the marginal area between healthy and plate, inoculated into an Erlenmeyer tions, spore carryover in sprayer will diseased tissue. Excisions should be flask containing sucrose-V8 broth, and be insignificant. Each treatment (e.g. made with a clean pair of scissors and placed on a shaker. Following sporula- titer) is applied to five plants and the specimens handled with forceps to tion, conidia are titered and healthy experiment conducted three times. lessen chances of additional contami- jointvetch seedlings infected as de- Jointvetch seedlings are inoculated nation. Using sterile humidity plates is scribed previously. with spore suspensions of the fungus recommended, but not required. Hu- using a hand-held mist sprayer or an midity plates (with lids) are incubated artist's airbrush atomizer (15 psi). To at room temperature for 2-3 days until Results & Discussion lessen variability between spraying orange masses of spores appear within techniques of different students, plants disease lesions (Figure 2A). Typical This experiment is currently con- should be sprayed until near-runoff spores of C. gloeosporioidesare shown in ducted by students in the general mi- with inoculum. Inoculated seedlings Figure 2B. Plates (specimens) may be crobiology class offered at Ball State may be air-dried for approximately incubated in complete darkness; how- University. Some of the materials five minutes or placed into a humidity ever, optimal sporulation occurs with needed to conduct this activity may not chamber immediately after spraying. incubation under fluorescent lights be available in high school laboratories; To provide sufficient percent relative (Gro-LuxTM:CoolWhite, 1:1) adjusted nevertheless, this exercise is appropri- humidity for infection (approximately to a 14-hour photoperiod. ate for high school biology, AP biology,

576 THEAMERICAN BIOLOGY TEACHER, VOLUME 59, NO. 9, NOVEMBER/DECEMBER1997 junior college, and university level stu- animal model for demonstrating dents. Choosing spore titer as an exper- Koch's postulates in the classroom. The imental variable not only teaches the additional step of recording and ana- Have student how to prepare serial dilutions, lyzing differences in plant (above youheard about NABT's but also provides optimal spore con- ground) dry weights (e.g. biomass or % centration(s) for conducting Koch's biomass loss) between treatments will new uHication? postulates while at the same time dem- introduce students to the importance of onstrating the inoculum density vs. using a computer as a scientific tool. If disease severity relationship. You may a computer is available, data can be choose to investigate different variables added to graphics and basic statistical including the effects of temperature, analysis software for additional class hours of leaf wetness, surfactant type activity. Hopefully your students will or concentration, and host specificity or have as much fun as ours do in proving age on C. gloeosporioides infection of Koch's postulates! northern jointvetch. Methods of disease control might be tested with vari- Watchfor the arrivalof ous systemic or protectant fungicides. An ancillary exercise could involve the References student calculating the "mathematical Anderson, R.N. & Walker, L.H. (1985). tud ent Downloaded from http://online.ucpress.edu/abt/article-pdf/59/9/574/48133/4450385.pdf by guest on 29 September 2021 factor" when determining spore titer Colletotrichum coccodes: A pathogen from different sized cubes on the hema- of eastern black nightshade (Solanum cytometer. ptycanthum). Weed Science, 33, 902- Research This exercise is not limited to just 905. northern jointvetch. Many other plants Charudattan, R. & Walker, H.L. (1982). could be used just as easily to demon- Biological Control of Weeds with Plant La oratories strate Koch's postulates. Plant patholo- Pathogens. New York: John Wiley & gists located at the state land-grant Sons. Partialsupport forthis work was provided by the National college or agricultural experiment sta- Daniel, J.T., Templeton, G.E., Smith, 5cienceFoundation through grant ESI # 9155206. tion in your area might be able to R.J., Jr. & Fox, W.T. (1973). Biological provide an alternative plant patho- control of northern jointvetch in rice genic microbe and host for conducting with an endemic fungal disease. this experiment. Weed Science, 21, 303-307. Sporulation of C. gloeosporioides oc- Joye, G.F. (1990). Biocontrol of Hydrilla curs within 5-7 days in shake cultures verticillata with the endemic fungus and is easily detected by the medium Macrophomina phaseolina. Plant Dis- turning orange in color. A single flask ease, 74, 1035-1036. containing 50 ml medium should pro- Kirkpatrick, T.L., Templeton, G.E., duce a titer of approximately 2 x 107 to TeBeest, D.O. & Smith, R.J.,Jr. (1982). _ Z _~~~~~~~~~~~~ Potential of Colletotrichum malvarum 1 x 108 spores/ml, more than sufficient for biological control of prickly sida. inoculum for this experiment. Several Plant Disease, 66, 323-325. flasks should be inoculated per chance Lorenzi, H.J. & Jeffrey, L.S. (1987). sporulation fails in one. Sporulation Weeds of the United States and their may be enhanced by removing flasks Control. New York: Van Nostrand m_'i from shaker daily and swirling con- Reinhold. * 0 . . tents to dislodge any growth stuck to Mitchell, J.K. (1988). Gibbago trianthe- 60 sides of vessel. If a shaker is not avail- mae, a recently described hyphomy- able, this fungus will also sporulate cete with bioherbicide potential for (albeit less than shake cultures) on control of Horse Purslane (Tri- * 0 0 0 _ PDA plates. To collect spores from anthema portulacastrum). Plant Dis- 00 PDA cultures, add chilled surfactant ease, 72, 354-355. solution to each plate and scrape sur- Mitchell, J.K. (1993a). Potential of Col- 000 face of medium with an inoculating letotrichum graminicola and Gloeocer- 3 E_0 loop to suspend spores. Decant sus- cosporasorghi as biological herbicides . pensions into a beaker and filter as for control of Johnson grass. Trends 0__ described previously. in Agricultural Sciences, 1, 31-36. Disease symptoms appear in in- Mitchell, J.K. (1993b). Influence of tem- fected plants within 4-7 days after perature and wetness duration on removal from the humidity chamber. Johnson grass control with Colletotri- 0 . Z00. chum graminicola and Gloeocercospora Death is observed in seedlings inocu- sorghi. Trends in Agricultural Sciences, 0 lated with spore concentrations greater 1, 37-44. 3_~~ than 1 x 104 spores/ml. Plants inocu- Prescott, L.M., Harley, J.P. & Klein, lated with titers ranging between 1 x D.A. (1996). Microbiology. Dubuque, _~~I 103 to 1 x 104 spores/ml exhibit typical IA: Wm. C. Brown Publishers. localized lesions and prove more suc- Riddle, G.E., Burpee, L.L. & Boland, cessful in demonstrating Koch's postu- G.J. (1991). Virulence of Sclerotinia n_ lates. sclerotiorum and S. minor on Dande- The COLLEGO-northern jointvetch lion (Taraxacum officinale). Weed Sci- interaction provides an excellent non- ence, 39, 109-118. Emil

FUN MICROBIOLOGY577