Popcorn and Sorghum Studies by USDA “Ag Lab” in 2013

dpf0901s CIIGA Havana IL 2009-02-05.ppt Slide #1

Popcorn and Sorghum Studies by USDA “Ag Lab” in 2013

Popcorn and Sorghum Studies by USDA “Ag Lab” in 2013

P. F. Dowd, E.T. Johnson, and

S.E. Sattler

P. F. Dowd, E.T. Johnson, and

S.E. Sattler

USDA / ARS, Peoria, IL and USDA/ARS, Lincoln, NE

USDA / ARS, Peoria, IL and USDA/ARS, Lincoln, NE

Presented at the CIIGA Irrigation Clinic, Havana, IL, February 13, 2014Presented at the CIIGA Irrigation Clinic, Havana, IL, February 13, 2014


Corn Borer Damage Colonized by Fusarium spp.Corn Borer Damage Colonized by Fusarium spp.


Brief List of Levels ToleratedBrief List of Levels Tolerated

FDA regulated levels of mycotoxins for human food materials:

Aflatoxinsaction threshold

20 ppb

Fumonisinsguidance level

2–4 ppm(depending on use)

Deoxynivalenol (DON)advisory level

1 ppm

FDA regulated levels of mycotoxins for human food materials:

Aflatoxinsaction threshold

20 ppb

Fumonisinsguidance level

2–4 ppm(depending on use)

Deoxynivalenol (DON)advisory level

1 ppm


Popcorn Corn Borer Damage Colonized by Aspergillus flavus

Popcorn Corn Borer Damage Colonized by Aspergillus flavus



Pest Resistance Gene Expression of Milk Stage Popcorn in Different Years

Two locations – Manito and Forest City

Three years 2010, 2011, 2012

Undamaged and insect damaged ears

Relate expression of genes to weather and mycotoxin levels



Gene Expression Results

Thousands of genes affected, including dozens of resistance genes

Some gene expression changed by as much as 500x

Many affected resistance genes interfere with protein digestion in molds and insects


Environmental effects on gene expression

Resistance gene expression was generally reduced in 2011 and 2012 compared to 2010

Some variation from location to location

Heat and drought (when power interruptions occurred) tended to reduce gene expression

Generally the same genes were affected whether ears were insect damaged or not

Insect damage increased expression of both insect and mold resistance genes in some cases.


Gene expression relationship to myctoxins

Multiple regression statistical analysis was used to examine effects of different combinations of resistance genes to find the ones most closely associated with mycotoxin levels in different years – limited to those that varied in expression in at least two years.

Mycotoxin levels were often associated with changes in expression of specific genes or gene combinations, sometimes the same ones

Several of those genes whose expression was associated with mycotoxin levels code for proteins that inhibit protein digestion in insects and/or molds


Caterpillar Feeding Responses to Sorghum Leaves with Altered Lignin Levels

Patrick F. Dowd, (USDA-ARS, NCAUR, Crop Bioprotection Research Unit, Peoria, IL)Jeff Pedersen and Scott Sattler (USDA-ARS, Grain, Forage and Bioenergy Research Unit, Lincoln, NE)

Results. Caterpillars fed diets containing relevant concentrations and proportions of phenolics did not show any significant differences in survivor weights from one another, although they were significantly smaller from those fed solvent control diets (data not shown). Generally feeding rates, mortality or survivor weights for either corn earworms or fall armyworms fed on different sized leaves of the mutant bmr6 and bmr12 lines did not indicate greater susceptibility compared to those noted for the wild type 623. However, there were several cases where the leaves of mutant lines were slightly, but significantly more resistant to one or both of the insect species, based on reduced feeding rates and /or smaller size of survivors. The pith of the stalks of the bmr6 and bmr12 lines caused significantly higher mortality of corn earworms and fall armyworms compared to the wild type plant pith. Field observations indicated feeding damage by chewing insects (presumably Japanese beetles base on their presence in adjacent corn plants and shape of holes), and some aphids were also present in July. Very little chewing insect damage was noted on the tillers examined in September, but aphids were more widespread and in some cases very abundant under leaf sheaths.

Introduction: Production of bioenergy from biomass promises to help satisfy energyneeds in a sustainable manner. One of the impediments to production of energy byfermenting biomass is the presence of lignin (1). Reduction of lignin levels through breeding or genetic engineering is possible, but may interfere with pest resistance wherelignin is a major component of resistance. Mutant lines of many plants exist which have altered and/or lower lignin composition. Some examples are the bm (brown midrib)mutants of maize, which can have increased stalk breakage when insect or diseaseoccurs (2). However, other bm grass species are acceptable agronomically (3). Sorghumalso has analogous mutants such as bmr6 (disrupted cinnamyl alcohol dehydrogenase = CAD) and bmr12 (disrupted catechol O-methyl transferase =COMT) (4).These lines have less lignin and are easier to ferment (1), but have not been examined forpest resistance. Different stages and tissues of the two mutants were examined for resistance to first instar corn earworms and fall armyworms. Field observations were also recorded.

Materials and Methods. Line Tx623 and lines containing the bmr6 and bmr12 allele were crossed and then backcrossed for 4 generations to yield stable near isogenic inbreds. Plants were grown in pots or outdoors under conditions described previously (5,6). Leaf sections from young and old plants, and stem sections from field grown plants were used in bioassays with first instars. Feeding rates (for leaves) and mortality and weights of survivors were determined as described previously (5). Field grown plants were also rated for mm2 of leaf damage on the main (July) and first tiller (September) stalks. The same composition and proportions of the phenolics (ferulic acid, coumaric acid, sinapic acid, vanillic acid syringyl acid and adipic acid) reported for the respective lines (4) were incorporated in diet fed both species of insects. Statistical differences were determined using Chi square (mortality) or analysis of variance (feeding rates and survivor weights) with SAS for Windows Version 8.0.

Disclaimer: The mention of firm names or trade products does not imply that they are endorsed or recommended by the USDA over other firms or similar products not mentioned. USDA is an equal opportunity provider and employer.

Acknowledgements: We thank A. Cranford, Z. Demcovitch, and D. Lee for technical assistance. This work was supported by Agricultural and Food Research Institute Competitive Grant #2011-67009-30026 from the USDA National Institute of Food and Agriculture and base funding to Agricultural Research Service CRIS projects.

References

Discussion. We did not observe any consistent increased susceptibility of the bmr lines to the insects we tested in the present study. Conversely, we did note several instances where resistance to insects in the lab and field was greater for the bmr lines compared to the wild type, especially bmr6. Variations in these trends may have been due to environmental or plant age influenced factors that were varying in the different plant stages examined. Genotype and environment can both influence the quality of agronomic traits in brown midrib lines (7). The high resistance of the pith of the bmr lines was somewhat unexpected. Considering that factors in the pith would likely be occurring in the leaf veins as well (based on similar colors) it is possible that the same components may be contributing to greater resistance of the bmr line leaves sometimes noted. Because in several cases there was a lower feeding rate to weight ratio for the bmr lines relative to the wild type when feeeding rates were equal or greater for the bmr lines, the bmr leaves may be less nutritious, and increased feeding relative to weights may be an indication of compensatory feeding.

Based on the different colors noted in the pith of the bmr6 vs. bmr12, the different location in the lignin biosynthetic pathway of the mutations may be responsible for producing different colored components. However, the very similar degree of mortality noted with both corn earworms and fall armyworms relative to one another for both bmr6 and bmr12 pith suggests a common factor is involved that is not related to the color difference. This resistance may be the result of accumulation of secondary metabolites from altered pathways, but does not appear to be strongly related to the altered levels of phenolics between the two lines and wild type plants. Alternatively, the changes in monolignols could be indirectly influencing expression of resistance genes unrelated to the secondary metabolites. Chemical and molecular analysis of the potential resistance factors in the bmr lines are in progress.

Future plans include the examination of transgenic sorghum lines with enhanced lignin levels for differences in insect resistance. Overall the present study suggests it may be possible to reduce lignin levels in sorghum, making them more suitable for fermentative production of ethanol without increasing insect susceptibility, although increased damage of younger leaves of bmr lines may be an issue.

1. Dien et al. 2009, BioEnergy Res. 2: 153-164.2. Barriere and Agillier 1993. Agronomie 13: 865-876. 3. Pedersen et al. 2005. Crop Science. 45: 812-819.4. Bout and Vermerris. 2003. Mol. Genet.Genomics 269: 205-214; Saballos et al. 2009. Genetics. 181: 783- 795.; Sattler et al. 2009. Plant Physiol. 150: 584-595. 5. Dowd et al. 2007. J. Agri. Food Chem. 55: 3421-3428.6. Dowd and White 2002. J. Econ. Entomol. 95: 628-634.7. Cassler et al. 2003. Crop. Science 43: 782-789 Palmer et al. 2008. Planta 229: 113-127

Table 2 Sorghum pith effects on corn earworms and fall armyworms ------------------------------------------------------------------------------------------

Corn earworms Fall armyworms Year %Mortality %Mortality------------------------------------------------------------------------------------------Field grownWild type 5.6a 22.2abmr6 51.3b 64.7bbmr12 36.6b 63.6b Lab grownWild type 1.7a 10.4a

bmr6 36.4b 27.7bbmr12 29.9b 25.5b------------------------------------------------------------------------------------------See Table 1 for legend. Pith from at least 8 plants of each type used.

Table 1. Sorghum leaf effects on corn earworms and fall armyworms------------------------------------------------------------------------------------------

Corn earworm Fall armyworm Feeding Weight Feeding Weight-----------------------------------------------------------------------------------------10 leaf plant, mature leaf (Greenhouse)Wild type 49a 0.23a 111a 0.53abmr6 53a 0.19b 137b 0.47bbmr12 54a 0.19b 126b 0.48b5 leaf plant, immature leaf (Plant growth room)Wild type 39a 0.21a 68a 0.35abmr6 48b 0.23a 54b 0.31bbmr12 50b 0.23a 70a 0.34a12 leaf plant, mature leaf (Plant growth room)Wild type 48a 0.24a 59a 0.37abmr6 41b 0.21 b 44b 0.42bbmr12 43ab 0.20 b 59a 0.34c12 leaf plant, mature leaf – recut (Plant growth room)Wild type 56a 0.23a 71a 0.42abmr6 45b 0.21ab 58b 0.36bbmr12 41b 0.20 b 61ab 0.38ab12 leaf, mature leaf (Field grown)Wild type 20a 0.13a 36a 0.35abmr6 19a 0.11 a 35a 0.36abmr12 17a 0.19 b 37a 0.35a----------------------------------------------------------------------------------------At least 10 leaves of each line were used. Mean values reportedare in mm2 (feeding) and mg (weights). Values followed by differentletters are significantly different at P< 0.05.

Table 3. Insect damage on field grown sorghum plants---------------------------------------------------------------------------------

Chewing Sucking% Incidence Damage % incidence

---------------------------------------------------------------------------------First stalkWild type 47.1a 7.3a 0.0a bmr6 20.8b 3.0a 0.0abmr12 46.7a 5.8a 7.1a

First tiller Wild type 31.2a 0.9a 25.0abmr6 18.2a 0.2a 13.6aBmr12 23.1a 0.4a 38.5a----------------------------------------------------------------------------------See Table 1 for legend. At least 12 plants of each type examined.





Introduction: Production of bioenergy from biomass promises to help satisfy energy

needs in a Wild tyesustainable manner. One of the impediments to production of energy byfermenting biomass is the presence of lignin (1). Reduction of lignin levels through breeding or genetic engineering is possible, but may interfere with pest resistance wherelignin is a major component of resistance. Mutant lines of many plants exist which have altered and/or lower lignin composition. Some examples are the bm (brown midrib)mutants of maize, which can have increased stalk breakage when insect or diseaseoccurs (2). However, other bm grass species are acceptable agronomically (3). Sorghumalso has analogous mutants such as bmr6 (disrupted cinnamyl alcohol dehydrogenase = CAD) and bmr12 (disrupted catechol O-methyl transferase =COMT) (4).These lines have less lignin and are easier to ferment (1), but have not been examined forpest resistance. Different stages and tissues of the two mutants were examined for resistance to first instar corn earworms and fall armyworms. Field observations were also recorded.




References

Discussion. We did not observe any consistent increased susceptibility of the bmr lines to the insects we tested in the present study. Conversely, we did note several instances where resistance to insects in th lab and field was greater for the bmr lines compared to the wild type, especially bmr6. Variations in these trends may have been due to environmental or plant age influenced factors that were varying in the different plant stages examined. Genotype and environment can both influence the quality of agronomic trats in brown midrib lines (7). The high resistance of the pith of the bmr lines was somewhat unexpected. Considering that factors in the pith would likely be occurring in the leaf veins as well (based on similar colors) it is possible that the same components may be contributing to greater resistance of the bmr line leaves sometimes noted. Because in several cases there was a lower feeding rate to weight ratio for the bmr lines relative to the wild type when feeeding rates were equal or greater for the bmr lines, the bmr leaves may be less nutritious, and increased feeding relative to weights may be an indication of compensatory feeding.













Wild type bmr6 bmr12





Introduction: Production of bioenergy from biomass promises to help satisfy energyneeds in a sustainable manner. One of the impediments to production of energy byfermenting biomass is the presence of lignin (1). Reduction of lignin levels through breeding or genetic engineering is possible, but may interfere with pest resistance wherelignin is a major component of resistance. Mutant lines of many plants exist which have altered and/or lower lignin composition. Some examples are the bm (brown midrib)mutants of maize, which can have increased stalk breakage when insect or diseaseoccurs (2). However, other bm grass species are acceptable agronomically (3). Sorghumalso has analogous mutants such as bmr6 (disrupted cinnamyl alcohol dehydrogenase = CAD) and bmr12 (disrupted catechol O-methyl transferase =COMT) (4).These lines have less lignin and are easier to ferment (1), but have not been examined forpest resistance. Different stages and tissues of the two mutants were examined for resistance to first instar corn earworms and fall armyworms. Field observations were also recorded.




References

Discussion. We did not observe any consistent increased susceptibility of the bmr lines to the insects we tested in the present study. Conversely, we did note several instances where resistance to insects in the lab and field was greater for the bmr lines compared to the wild type, especially bmr6. Variations in these trends may have been due to environmental or plant age influenced factors that were varying in the different plant stages examined. Genotype and environment can both influence the quality of agronomic traits in brown midrib lines (7). The high resistance of the pith of the bmr lines was somewhat unexpected. Considering that factors in the pith would likely be occurring in the leaf veins as well (based on similar colors) it is possible that the same components may be contributing to greater resistance of the bmr line leaves sometimes noted. Because in several cases there was a lower feeding rate to weight ratio for the bmr lines relative to the wild type when feeeding rates were equal or greater for the bmr lines, the bmr leaves may be less nutritious, and increased feeding relative to weights may be an indication of compensatory feeding.














Potential Bioenergy Sorghum Lines (NIFA funded study)

Laboratory studies under controlled conditions have generally indicated no increase in amounts of damage of the 4th leaf from 5 leaf plants of low compared to normal lignin sorghum isolines by caterpillars.

Laboratory studies under controlled conditions have generally indicated reduced leaf damage, especially for bmr6 plants for the 7th leaf of 12 leaf plants by caterpillars.

Laboratory assays under controlled conditions have indicated higher mortality of caterpillars fed on pith from the bmr lines compared to normal lignin type when fed pith removed from between the uppermost and second leaves at grain fill.


Caterpillar leaf damage to sorghum in 2013% Incidence Damage - cm2

8-16-13

Normal lignin 4.2a 26.7+/-2.5a

Bmr6 1.8b 13.1+/-1.3b

Bmr12 5.5a 22.4+/-1.3a

9-3-13


Bmr6 2.6b 12.4+/-1.0b

bmr12 5.9a 24.6+/-1.4c

9-25-13


Bmr6 2.3b 9.3+/-0.9b

Bmr12 6.8c 17.1+/-1.2a


Stalk boring damage to sorghum in 2012 and 2013

% Incidence Length in cm

9-25-12

Normal lignin 19.7a 4.8a

Bmr6 22.3a 2.8b

Bmr12 21.1a 3.2b

9-25-13

Normal lignin 0.3a 0.6a 8.8

Bmr6 0.2a 0.2a 3.0

Bmr12 1.5b 1.4b 7.0




Grasshopper leaf damage to sorghum in 2013

% Incidence Damage - cm2

9-3-13


Bmr6 2.9b 36.4+/-2.6a

bmr12 4.6c 34.2+/-2.3a

9-25-13


Bmr6 0.8b 50.8+/-8.2a

Bmr12 4.6a 49.4+/-8.7a


Aphid presence in sorghum in 2013

% Incidence Rating 1-4 scale

9-3-13


Bmr6 5.6a 1.1+/-0.02b

bmr12 11.2b 1.3+/-0.05a

9-25-13 stem sections


Bmr6 16.8b 1.9+/-0.1a

Bmr12 20.0b 1.9+/-0.1a


Disease lesion leaf damage to sorghum in 2013

% Incidence Damage - mm2

8-16-13


Bmr6 1.9b 5.8+/-0.9a

Bmr12 6.0c 6.2+/-0.6a

9-3-13


Bmr6 0.24a 2.4+/-0.5a

Bmr12 0.15a 2.0+/-1.0a


Summary of low lignin sorghum studies near Havana in 2013

Caterpillar, aphid, grasshopper and disease lesion damage were noted

Low lignin lines were generally damaged no worse than the normal lignin sorghum

Low lignin line bmr6 often had less caterpillar damage than the normal lignin and bmr12 line

Results were similar to those found in 2012

Results were similar to what has been noted in laboratory and small plot assays


Documents

Popcorn and Sorghum Studies by USDA “Ag Lab” in 2013