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Entomophaga

, Volume 20, Issue 3, pp 229–240 | Cite as

Large-scale production of the fungal pathogenHirsutella thompsonii in submerged culture and its formulation for application in the field

  • C. W. McCoy
  • A. J. Hill
  • R. F. Kanavel
Article

Abstract

A large-scale method for producing the fungal pathogen,Hirsutella thompsonii, in submerged culture was developed in the laboratory to supply adequate quantities for studies of field efficacy against the citrus rust mite. Laboratory fermentors consisted of 18.9 liter sterilizable solution bottles stopped with aeration heads that were connected in series to supply a closed system for the passage of the sterile compressed air necessary for agitation and for supplying oxygen to the liquid enrichment.

The medium contained dextrose at 5 mg/ml, yeast extract and peptone at 5 mg/ml and 0.5 mg/ml, respectively, and essential mineral salts. Dow Corning antifoam V-30 emulsion, and tetracycline hydrochloride (980 mg/g) were included at optimum concentrations of 25 ppm and 5 mg/100 ml, respectively. Each vessel contained 12 liters of media and was inoculated with 75 ml of fragmented mycelia. At 22–26°C, an average of 400 grams (wet wt.) mycelia per vessel were produced after a 96-hr incubation.

Upon completion of the fermentation process, mycelia were recovered from the liquid end-products via filtration and stored as mat-mycelia in stainless steel holding pans at 10°C. The mat-mycelia appeared to lose viability some time after 64 days in storage.

The sporulation phase of pathogen production was excluded, and the fungus was applied as mycelial fragments by taking the mat from cold storage and formulating it on the day of application. Five hundred gram quantities of mat-mycelia were blended in 500 ml of water for 2 min to form a slurry and then transferred to 18.9 liter solution bottles for transport to the field. All spray formulations (different additives used as spreader stickers and protectants for the mycelia against heat or desiccation) were added and mixed directly in the tank of the sprayer. Formulations containing unsulphured molasses or citrus molasses in combination with Dacagin performed most effectively in field tests.

No skin irritation, inhalation difficulties, or symptoms of pathogenicity were experienced by laboratory personnel as a result of exposure toH. thompsonii during the 3-year production effort.

Keywords

Submerged Culture Tetracycline Hydrochloride Laboratory Fermentor Soufre Sporulation Phase 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Résumé

Une méthode de production en grande quantité deHirsutella thompsonii en milieu liquide a été mise au point au laboratoire pour fournir des quantités suffisantes à l'expérimentation de terrain contre l'acarien des Citrus. Les fermenteurs de laboratoire consistaient en bouteilles de 18,9 litres stérilisables fermées par des tubulures connectées en séries assurant l'alimentation en air comprimé nécessaire pour l'agitation du milieu et pour l'apport d'oxygène.

Le milieu est constitué de 5 mg/ml de dextrose et autant d'extrait de levure, de 0,5 mg/ml de peptone et des sels minéraux essentiels. L'émulsion antimousse Dow Corning V-30 et l'hydrochlorure de tetracycline (980 mg/ml) sont ajoutés, aux concentrations optimum de 25 ppm et 5 mg/100 ml, respectivement. Chaque récipient renferme 12 litres de milieu et est inoculé avec 75 ml de fragments mycéliens. A 22–26°C, on obtient après 96 heures d'incubation une moyenne de 400 grammes en poids humide de mycélium.

Ce mycélium est extrait par filtration et conservé à 10°C dans des récipients en acier sans étain. Ce tapis de mycélium perd sa viabilité au bout de 64 jours de conservation.

Le champignon a été utilisé pour les applications sous forme de fragments mycéliens en prenant le tapis conservé au froid et en le formulant le jour de son emploi. 500 grammes de ce «tapis mycélien» ont été mis dans 500 ml d'eau pendant 2 minutes pour obtenir une bouillie transférée dans des bouteilles de 18,9 litres pour le transport sur le terrain.

Toutes les formulations pour la pulvérisation (divers adjuvants pour l'adhésion et pour la protection des mycéliums contre la chaleur et la dessication) ont été effectuées directement dans le réservoir du pulvérisateur. Des formulations renfermant des mélasses sans soufre ou des mélasses de Citrus en mélange avec du Dacagin ont donné les meilleurs résultats dans les essais en champ.

Aucune irritation de la peau, aucun inconvénient à la suite de l'inhalation, ni symptômes de pathogénie n'ont été constatés chez le personnel du laboratoire à la suite du contact avecH. thompsonii pendant les 3 ans d'étude de cette production.

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Copyright information

© Le François 1975

Authors and Affiliations

  • C. W. McCoy
    • 1
  • A. J. Hill
    • 1
  • R. F. Kanavel
    • 1
  1. 1.U.S. Horticultural Research Laboratory, Agricultural Research ServiceU.S.D.A.OrlandoU.S.A.

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