the Symposium on Wheats for More Tropical Environments - cimmyt

1'70an extremely high incidence of naturalscab infection. However. by takingadvantage of both natural andartificially inoculated fusarium. bothlocations are excellent sites for scabscreening (1).Fusarium i_IatioDaDd culture preparationFusarium-infected wheat spikes. forpure spore isolation and fresh inoculumpreparation. were collected from Tolucaand Pat.zcuaro. Spore isolation andpuriflcation were made on corn mealagar (CMA) (2) in <strong>the</strong> laboratory at EIBatan. For indiVidual-spike and massinoculation (spikes in one-meter singlerows). a large volume of fusariumisolates were multiplied on CMA andmungbean extract (W.Y. Zhong andW.S. Jun. personal communication).PreparatioD offUNJ'ium .pore inoculumTo prepare <strong>the</strong> fusarium inoculum.cultures grown on CMA are placed in adisinfected food blender. and blended forone minute to make a thin puree. TheCMA-culture puree is <strong>the</strong>n passedthrough several layers ofsterilized cheesecloth to obtain a clear filtrate. Whenmungbean extract is used as <strong>the</strong> culturemedium. <strong>the</strong> culture is blended for only afew seconds and filtered as above. In bothcases. <strong>the</strong> concentrated clear filtrate is<strong>the</strong>n diluted with sterilized distllled wateruntil a desired spore count per cc ofwater is attained. Thirty to fifty thousandspores per cc ofwater are suitable forCIMMYT conditions.lDoculatioD meth0d8Two different inoculation methods weredeveloped and modified at CIMMYT forcontrolled scab screening. Thesemethods are suitable both in <strong>the</strong>greenhouse and in <strong>the</strong> field underMexican conditions. They are:• The cotton method-With thismethod. a tiny tuft of cotton (cottonswab for laboratory and hospital use)is penneated with <strong>the</strong> distilled watercontaining fusarium spores insuspension. One-fourth of <strong>the</strong> cottonswab. <strong>the</strong> size ofan outer wheatglume. is pushed between <strong>the</strong>glumes and placed in contact with<strong>the</strong> an<strong>the</strong>r in a wheat floret. Sharppointedtweezers of <strong>the</strong> kindgenerally used for emasculationpurposes are used for separating <strong>the</strong>cotton tufts and for inoculating. Aglassine bag is <strong>the</strong>n placed over <strong>the</strong>inoculated wheat head to preventdamage by wind. dust or excesshumidity (rain or dew). This cottonmethod is <strong>the</strong> most precise forcontrolled scab study in <strong>the</strong>greenhouse and in <strong>the</strong> field.The method is comparable to that ofinoculation by injection with asyringe. However. unlike <strong>the</strong>injection method. <strong>the</strong> cotton methodplaces <strong>the</strong> inoculum at a specifiedinoculation point without injuringplant tissue. With <strong>the</strong> syringemethod. <strong>the</strong> water suspension candrip. contaminating o<strong>the</strong>r parts of<strong>the</strong> spike. Also <strong>the</strong>re is <strong>the</strong> possibllityof applying less inoculum withsyringe applications because. when<strong>the</strong> needle is pushed through <strong>the</strong>spike. a portion of <strong>the</strong> inoculum maybe discharged into <strong>the</strong> air. Theneedle also injures <strong>the</strong> wheat tissue.• The spray method-With thismethod. fusarium spores suspendedin water are sprayed on wheat heads.using various types and sizes of handgarden sprayers with various nozzleattachments. Also. a back-packmotor-driven sprayer can be used.For CIMMYT's advanced wheat linescreening study. a small handatomizer (commonly used as agarden pesticide sprayer). with ameter long. hard-walled plastic tube.is used. Inoculum is placed in oneliterplastic containers. and <strong>the</strong> tubefrom <strong>the</strong> spray nozzle is immersed in<strong>the</strong> container.