1. Mass production of entomopathogenic fungi: challenges to the ‘mycodream’
Stefan Jaronski
USDA ARS NPARL, Sidney MT

2. A History of Mycoinsecticide Development
First fungus (Metarhizium) mass-produced and used against insects in Russia.
1890’s - Beauveria released throughout entire Midwest.
1930’s - Beauveria experimentally mass-produced on grains, soybean mash in U.S.
Advent of chemical pesticides ...
1970’s & 1980’s - Fungi developed in China, USSR, Brazil, and Europe; Attempts in U.S. fail, except Abbott’s Mycar.
1990’s-2000’s - Fungi developed in Columbia, Cuba, Nicaragua, and Mexico, but on only ‘Mom & Pop’ scale. Beauveria, Metarhizium registered; commercialized in US; Beauveria in France.
And today (2013) …
The first attempts to “commercially” produce fungi to control insects were in Czarist Russia in the 1880s.

3. Faria and Wraight Biological Control 43 (2007) 237–256
Mycoinsecticides: active, commercial products in 2006
Faria and Wraight Biological Control 43 (2007) 237–256

4. Entomopathogenic Ascomycetes Meet the Current Players
Beauveria bassiana & brongniartii
Metarhizium “anisopliae” & acridum
Lecanicillium longisporium, & muscarium (the fungus formerly known as Verticillium lecanii)
Isaria fumosorosea (formerly known as Paecilomyces fumosoroseus)
© Koppert

5. (Think “fatal athlete’s foot” of insects)
Dispersion of Aerial Conidia
Germination & Penetration thru Cuticle
Proliferation thru Haemocoel as Blastospores or Hyphae
Transformation upon Insect’s Death
Sporulation
Typical Life Cycle
Regardless of species the life cycle is pretty much the same. [Describe]
Describe some of the photos…
(Think “fatal athlete’s foot” of insects)

6. Propagule types in the entomopathogenic Ascomycetes
Aerial Conidium
Barron, U Guelph
Blastospore
Microcycle Conidium
Jenkins & Prior 1993
Mycelium, microsclerotia
These fungi have four major propagule types that have been or can be used.
In nature the aerial conidium is the primary infectious propagule. These are the spores that are produced on the exterior of fungus-killed insects.
Blastospores are the proliferative stage INSIDE the insect for many of these fungi and can also be produced in liquid fermentation. (Not surprising when one considers an insect is merely a six-legged flask of culture medium..
Under certain liquid fermentation conditions, mainly inorganic nitrogen, Beauveria and Metarhizium can produce “microcycle” conidia as can be seen here The conidia are not true conidia and are produced on the ends of hyphal strands.
Lastly mycelium, the major form of fungal proliferation, or mycelial derivatives such as the microcycle conidia I talked about yesterday, can be used. But here the mycelium or its derivatives are really producers of conidia – a way to deliver conidia to the insect

7. These fungi are not fastidious . . .
Mass Production
These fungi are not fastidious . . .
“All You Need Is Love”
 Simple Carbon Nitrogen Sources Eg., Dextrose + KNO3  Oxygen  Moisture  Proper Temperature

8. Basic Mass Production Methods
Grain or Organic Carrier in Bags, Trays, Chambers
Nutrient-Impregnated Inert Carrier, in Bags, Trays or Chambers
Liquid Surface Culture (‘Plastic Pillow’)
Woven Fabric Belts/Fiber-Paper Sheets
Submerged Liquid Fermentation (Blastospores, Mycelium, Metarhizium Microsclerotia)
Conidia
There are a number of varied production methods to produce fungal spores …

9. Not all fungal isolates are created equal with the inalienable right to sporulate

11. And what about Metarhizium?
6.8x1012

12. Virulence vs. Spore production
Decisions, decisions …
Virulence vs. Spore production

13. Each point represents mean Kill Ratio Relative to BB1002 Std
Spore production vs. Virulence
2.5E+13
Each point represents mean
2E+13
of 2-3 bioassays
BB1067
1.5E+13
Yield (spores/Kg substrate)
SC19
SE7
SF3
BB1077
1E+13
BB1065
BB1076
SC15
SC18
SD4
BB1002
SD13
SF5
SA8
5E+12
SC14
BB1074
BB1071
MA5197
0.00
0.50
1.00
1.50
2.00
2.50
Kill Ratio Relative to BB1002 Std

14. A balance between field efficacy and production
Spore Production
# spores/ Kg or L 
Efficacy Spores/ha
Kg of Substrate or Liter Fermentation (=$$) per hectare of fungus

15. What are efficacious field rates???
1.25x1012 – 5x1013 spores / hectare (broadcast application)
WHY so many?
2.5x1013/ha =
2.5x105/cm2 (2500/mm2) on flat surface 6.2x104/cm2 (62/mm2) on foliage w LAI of 4
Beauveria GHA vs. DBM, LC95 = 1567 spores/mm2
Beauveria GHA vs. BAW, LC95 = 93,325 spores/mm2
M. acridum FI985 vs. Locusta migratoria, LD95 ~79,000 spores; w 6 cm2 area = 132 spores/mm2

16. The Basics -- Need vs. Abilities
Base Use Rate: (2.5x1013 conidia/ha/application)
Production System Units/ha/appl
Soviet sporulated mycelium m2 (8x1012/m2)
Czech Liquid Surface Culture (1x1014/m2) m2
Low tech Bag Solid Substrate (2x1012/Kg) Kg
Engineered Biphasic SSF Beauveria (2x1013/Kg) Kg
Engineered Biphasic SSF Metarhizium (6x1012/Kg) Kg
Submerged Blastospores Bb, Ifr (5x1011/L) L (if 1x1012/L as per Fargues et al.) L
But how are these systems able to handle production needs??
Here we have a comparison based on published data for the different methods.
The basic need is 2.5E13 conidia per hectare
I’ve broken down capacities of the different methods into production units per hectare
At this point only engineered biphasic solid substrate fermentation has the efficiency to meet commercial needs, even when differences in cycle time are considered.

17. It all boils down to … Mass Production:
$ Cost of fermentation ( per Liter or Kg) per hectare
as affected by total needed capacity
and capacity is driven by economics of the market

18. Some examples: treated needed Crop %mrkt acres spores capacity
1x1013/ac/app (2.5x1013)
LT SSF HT SSF LIQ Ferm 2x1012/kg 2x1013/Kg 2x1012/L
Some examples:
treated needed Crop %mrkt acres spores capacity
Greenhouse , x ,000 Kg (7,287 ha) ,100 Kg ,000 L
Sugar beets , x ,225,000 Kg (89,878 ha) ,800 Kg ,250,000 L
LTSSF = bag-based solid substrate fermentation
HTSSF = high tech (Mycotech) solid substrate fermentation
LF = liquid fermentation
NB:
Corn use rate is 1x1013 conidia/acre, based on Mycotech data
Sugarbeet use rate is 2x1013/acre because of data of my best Metarhizium
If the Hi tech SSF yielded only 1E13/Kg (e.g., typical Metarhizium production) then one needs 167,000 Kg capacity.
Corn , x ,500 Kg (280,567 ha) 3,465,000 Kg ,930,000 L

19. And then there are the fermentation “containers”
Need
2.5 Kg bags
10,000Kg batch
10,000 L ferm. runs
Greenhouse (7,287 ha. or 50% market)
Lo Tech SSF
91,000 Kg
36,400!
Hi Tech SSF
9,100 Kg
0.9
Liquid Ferm.
182,000 L
18.2

20.  Production High tech vs. low tech (decisions, decisions, decisions)
High tech Low labor
Low tech High labor
$1 MM plant w/ 30 people ? 
Tradeoffs:
capital cost (one time) vs. labor costs (year after year).
$41,600 is generally little in the U.S. A garbageman on the East Coast can draw $40,000-50,000 plus 30% benefits! On the other hand the median salary in Butte MT was $19,000, because it is a depressed economy. A GS-5 level technician in USDA makes $44,000 while GS-7 makes 55,000
Payroll = $1,500,000
$5-8MM plant w/ 5 people ?
Mean salary+benefits $50,000
Payroll = $250,000

21. Mass production is possible, is a fact
Areas of low wages, smaller, local needs:

22. Efficient, cost effective high output production system
IS possible
1 run = 660 Kg Bb spores ( x1011/g) 8x1016
3, E13/ha

23. The Pieces of the Commercial Puzzle
NTO
Safety
Spore
Production
Shelf Life
Virulence
Human
H20 UV
Ecology
Temp.

24. Quality Mass Production as well as Quantity Percent recovery
Purity (high & consistent)
Initial viability
Genetic stability
Consistent Infectivity/virulence
Shelf life

25. Recovery:
In production, as in home finance, “It’s not how much you make, as much as how much you keep by the end of the month…”

26. Purity The effect of varying TGAI titers: TGAI Grams TGAI Grams Inerts
1.0E11/g
200
254
2E13 spores/lb. WP
1.5E11/g
133
321

27. Purity Initial viability: % viability % tgai Microbial contaminants:
< 5 logs of a.i. e.g., < 4.4E5/g for 2E13 /lb. NO Shigella, Vibrio, Salmonella, other enterics

28. There is great genetic variation among fungus isolates
(Beauveria isolates from grasshoppers at 1 location, on 1 day)

29. Considerable Genetic Plasticity
Beauveria can be genetically diverse from one host group – grasshoppers -- in one region (MT-ND), even one site (AFLP EcoR1-AAC, -AGG Jaronski & Kaufmann, unpubl)
Considerable Genetic Plasticity

30. The “nightmare”
“Fungi are notorious for losing virulence and changing their morphology when successively subcultured on artificial media.”
Butt et al 2006 Degeneration of Entomogenous Fungi
Wang, Butt, St. Leger Microbiology 151: 3223–3236
Jaronski

31. Genetic stability during production
660 Kg
= 8x1016
1 spore
Slants = 5x108
15 L = 7.5x1012
1500 L = 1.5x1014
1x108 –fold increase in cells
8x1016 –fold increase in cells

32. Maintaining Genetic Stability
Choose the “right” species and isolate
Minimize in-vitro culturing of strain
Maintain active program of strain passage through target insects
Monitor, monitor, monitor - shelf life - virulence - molecular id

33. RFLP profiles of Beauveria bassiana GHA vs other strains

35. Shelf life (<20% loss viability, 1+ year at 20-25 C)
is affected by
Moisture
Temperature
Strain Differences
Nutrition & Fermentation Conditions
Formulation

38. 708 days
510 days
183 days

39. Packaging can greatly affect product stability
Hard Sided vs. Flexible Packages
Plastic vs. Foil
Packaging Breathability
Oxygen Transmission Rate
Water Vapor Transmission Rates

40. Solutions?  Lower efficacious use rate …
Create a transgenic SUPERFUNGUS! Kills faster with lower dose and jumps over tall buildings in a single bound
Better persistence thru ‘better chemistry’ (#1 UV protectants)

41. Manipulations during production to improve the spore
Solutions?
( Lower efficacious use rate …)
Manipulations during production to improve the spore

42. C:N ratio of medium could enhance infectivity, virulence
Tantalizing Glimpses of how the Medium could affect the Message (spore)
C:N ratio of medium could enhance infectivity, virulence
Water activity of medium can affect polyol content of spores, which germinate at lower humidity
Millet solid substrate supplemented with corn oil increased heat tolerance
Salicylic acid supplement can increase thermotolerance of spores
Conidia produced under some nutritional or osmotic stress had better UV and heat tolerance
Type of carbohydrate in substrate can increase thermotolerance of spores
Mn+2, Sucrose, Fe+3 supplementation can increase spore thermotolerance

43. Caveat emptor! Most of such studies done with agar media
In many cases spore yield was greatly reduced (too much?)
We still do not know how much improvement in UV, heat tolerance even virulence, improves field performance, esp. in ‘farmer’s hands

44. Solutions? Let’s consider better application approaches
Concentrating spores into key arena
In-field multiplication of fungus (sweet whey adjuvant, nutritive granules, microsclerotia)
Bring the insect to the fungus
Vector the fungus to target area (flowers) with bees
Inoculation to establish endophytic presence

45. how about IPM? (Where the fungi don’t have to do all the work)
And then,
how about IPM? (Where the fungi don’t have to do all the work)

46. Thank you