United States Department of Agriculture

Peg RedinbaughUSDA,ARS & Ohio State Univ.

Maize Lethal NecrosisPerspective from the U.S. Midwest

2

• Mark Jones

• Jane Todd

• Kristen Willie

• Chris Nacci

• Lucy Stewart

• KALRO-Anne Wangai, Bramwel Wanjala, James Karanja, Lilian Gichuru

• CIMMYT-George Mahuku, Dan Jeffers, Bish Das, B. Prasanna

• Venganza-Chuck Niblett

• TPRI-Mohamed Mpina

• RAB-Theodore Asiimwe, Daniel Niyikeza, JoviaKamatenisi

• NARO-Andrew Kiggundu, Godfrey Asea

• OAF-Thierry Mugwaneza

• José Zambrano

• David Francis

• Bryan Cassone

• Victoria Bulegeya

• Sally Miller

• Fulya Gurel

• Xing Ma

Thanks to –

• KEPHIS-Francis Mwatuni

• OkSU-Kay Scheets

• UMN-Ben Lockhart

• IPPE-Sevgan Subramanian

• WSU-Hanu Pappu

• Pioneer-Adriana Tomas

USDA FAS

L. striatellus

Maize Lethal Necrosis

Location Date Potyvirus

Peru 1973 NR

U.S. KS/NE 1976 WSMV

Argentina 1982

Thailand 1982

Mexico 1987

U.S. HI 1992 MDMV

China 2011 NR

Kenya 2012 SCMV

Rwanda 2013 SCMV

DRC 2013 SCMV

Taiwan 2014 SCMV

• SCMV is distributed world-wide,

including eastern Africa.

• MCMV emerged in the Eastern

Hemisphere since 2010.

• Factors involved in MCMV

emergence?

• Maize thrips

• Global seed movement??

• Seed transmission??

• Soil transmission??

L. striatellus

MLN Control

• Pathogens and Reservoirs– MCMV and a potyvirus

– Other viruses

– Weedy and crop reservoirs

– Continuous maize as a reservoir

– Maize seed as a reservoir

– Soil as a reservoir

• Susceptible hybrids and cultivars– Role of virus resistance and tolerance in control

• Vectors– MCMV: maize thrips vs. coleopteran insects

– Potyviruses: a number of aphid species

• Suitable environment– Environmental stress exacerbate MCMV to give MLN

Vectors

HostEnvironment

Pathogens

& Reservoirs

Discovery of MCMV and CLN in the U.S.

• In 1976, maize along the Republican River in Kansas

developed symptoms:

• Mosaic on leaves and husks

• Leaf necrosis progressing inward from the

margins

• Premature plant death

• Tassel necrosis

• Poor ear development, no seed set.

• Three viruses were present in samples:

• MCMV – similar to the Peru virus

• MDMV - Maize dwarf mosaic virus

• WSMV - Wheat streak mosaic virusNiblett & Claflin.1978. Plant Dis. Rep. 62:15-19.

Uyemoto. 1983. Plant Dis. 67: 7- 10.

Kansas

Potyviruses in OhioIncidence Relative Abundance

Sweet corn J-grass

MDMV-OH 195.8 3,579

SCMV-OH 2 29.5

SCMV-BD8 0.7 11.1

SrMV 1.7 30

JGMV 0 0.2

PenMV 0.5 8.4

Year MDMV SCMV

J-grass 2011 40/82

2012 20/33 1/33

Sweet corn 2011 15/26

2012 43/56 2/56

Maize 2011 3/11

• MCMV is NOT known in most of the U.S. Cornbelt.

• MDMV, SCMV and other potyviruses are present in OH.

• Incidence in maize lower than in sweet corn and Johnsongrass.

• Abundance is much lower in maize than in Johnsongrass.

Stewart et al. 2013. Phytopathology 104:1360-1369.

Kansas

L. striatellus

• Jardine http://www.plantpath.ksu.edu/pages/extension/factsheets• Uyemoto 1983. Plant Dis. 67: 7- 10.

• Stack (UNL) https://pdc.unl.edu/agriculturecrops/corn/maizechloroticmottlevirus

• Nelson et al. http://www.ctahr.hawaii.edu/oc/freepubs/pdf/PD-79.pdf

MCMV Control in the U.S.

MLN is an occasional and local problem. In the U.S. mainland it is found mostly in the Republican River Valley KS and NE.

Disease control measures include: • Winter

• Crop rotation

• Virus resistant/tolerant hybrids

• Weed control, especially Johnsongrass

• Diabrotica population control

MLN is a chronic problem in Hawaii.

Disease control measures include:• Weekly insecticide sprays to control thrips

vectors.

• Control of grassy weeds that serve as alternate hosts for virus and thrips

• Scout fields regularly and remove any symptomatic plants.

• Keep unnecessary people and machines out of the field to reduce mechanical transmission and spread of MCMV.

MCMV and SCMV in East Africa

• MCMV incidence high in

all three countries.

• SCMV incidence

significant, but lower.

• Antisera from two

sources behaved

differently in ELISA.

Location #Samples MCMV SCMV Both

W. Kenya 1297 967 (75) 539 (42) 466 (36)

Uganda 52 32 (62) 12 (23) 11 (21)

Tanzania 39 24 (62) 27 (69) 20 (51)

MCMV SCMV

Pos. both antisera 48 22

Neg. both antisera 2 26

Pos. A/Neg. B 28 16

Pos. B/Neg. A 3 17

Kansas, USA

Yunnan, China sugarcane

Yunnan, China

R4, Rwanda

R3, Rwanda

Sichuan,China

R1, Rwanda

Nebraska, USA

Bomet/Naivasha, Kenya

Yunnan2, China, sugarcane

R2, Rwanda

Yunnan3, China

NC 002598 PMV

KP851970 Naivasha Bomet, Kenya

MCMV Genome Phylogenies

The U.S. isolates of MCMV are distinct from

the Asian/East African isolates, but still highly

related. Sequence ID’s are >95%.

Variation among East African MCMV isolates

• CP amino acid sequence alignment• 97.0-100% identity• Serological and molecular diagnostics should be

straight forward

• 96.4 -100% sequence ID for coding & intergenic nucleotide sequences among MCMV contigs for Kenya/Uganda samples

KS Isolate

SCMV Diversity in Kenya/Uganda

• High variability among SCMV isolates. Within types 90-99% ID; between types <80-85% ID.

• Naivasha/Bomet samples had SCMV Rwanda and BD8 types.

• Kenya/Uganda survey samples had Rwanda-like, OH-like, BD8-like and China-like isolates.

• Most isolates are Rwanda-like

• Working on relative abundance and distribution.

SCMV CP variation and diagnostics

• Translated CP sequences have 85 –

100% ID, similar to the with whole

genome analysis.

• Most variation in N-terminus of CP.

• This is consistent with previous results

indicating high specificity of antibodies

made to the N-terminus of

potyviruses, and group-specificity of

antibodies to C-terminus

• SCMV sequence diversity is

consistent with the observed

differential responses of antisera.

Hints that other viruses are involved

• MYDV-RPV is in the genus Polerovirus, family Luteovirus. It is transmitted primarily by Rhopalosiphum maidis and infects primarily maize.

• A contig with 74% sequence ID to MYDV-RPV was identified from Naivasha/Bomet, Kenya.

• Similar sequences were identified from across Kenya/Uganda, but require confirmation and analysis of distribution.

• We have some indication that other potyviruses may be present, e.g. JGMV and SrMV. Further analysis of distribution and confirmation are required.

1000 2000 3000 4000 5000

Contig with 74% ID to MYDV-RPV

Maize yellow dwarf virus-RPV genome

The pathogen mix in East Africa

is likely more complex than in the

U.S. Cornbelt, but MCMV is the

major issue in both locations.

MCMV Reservoirs-Alternative Hosts

U.S. Cornbelt

• Bockelman et al.

– 19 Graminacous hosts identified by

inoculation with MCMV.

– MCMV was not found in 14 species

collected from field.

• Cultivar-dependent sorghum infection

(asymptomatic).

• WINTER means that maize is NOT a

reservoir.

Bockelman et al. 1982. Plant Dis. 66: 216-218.

East Africa

• Sorghum (S. bicolor), Proso millet

(Panicum miliaceum), finger millet

and foxtail Setaria italica became

infected after inoculation in Kenya.

• MCMV was detected in finger millet,

sorghum, sugarcane, Napier grass

(Pennisetum purpureum) and

Kikuyu grass (P. clandestinium)

collected in Kenya and Uganda.

• Maize is a significant reservoir.Alternative and maize hosts are a more likely source of viral

inoculum in East Africa.

Virus Transmission – Seed

• Bockelman et al. – no seed transmission from MCMV-

infected plants from 2153 seed from 14 inbred lines. Detection sensitivity ~0.2%.

– no seed transmission 1898 seed of 5 hybrids. Detection sensitivity of ~0.2%.

• Jensen et al. – 17/42,000 seed produced an

infected plant (0.04%).

– Transmission was not evenly distributed among lots. 16/17 infected plants came from one company.

Lot# #seed #Infected % Trans

5 3000 1 0.033

7 3000 10 0.333

8 3000 2 0.067

9 2000 4 0.200

21 others 31000 0 0

Bockelman et al. 1982. Plant Dis. 66: 216-218.

Jensen et a. 1991. Plant Dis. 75: 497-498.

Seed transmission is low, but

detectable. Data suggest

variation among genotypes.

MCMV Transmission by Insects

Nault et al. 1978. Phytopath. 68:1071-1074.

Jensen. 1985. Plant Dis. 86: 864-868.

Jiang et al. 1992. Crop Prot.. 11: 248-254

Cabanas et al. 2013. J. Econ. Ent. 106, 16-24.

Species Order # Insects % Trans.

Oulema melanopa Coleoptera 142 54.2

Chaetocnema pulicaria Coleoptera 215 5.2

Systena frontalis Coleoptera 175 2.4

Diabrotica virgifera Coleoptera 190 17

D. longicornis Coleoptera 95 13.9

D. undecimpunctata Coleoptera 105 15.6

Popillia japonica Coleoptera 55 0

Adoretus sinicus Coleptera 19 0

Agrotis ipsilon Lepidoptera 50 0

Aphids (4 species) Hemiptera 1860 0

Whiteflies (1 species) Hemiptera 240 0

Hoppers (5 species) Hemiptera 375 0

Tetranychus sp. Trombidiformes 24 0

Frankliniella williamsi Thysanoptera 10 100

Maize thrips Cereal leaf beetle

Diabrotica spp.

Crop Rotation and TillageIn Kansas/Nebraska

• In the 1960’s, two year moratoriums were imposed on growing maize. This reduced insect populations, and increased future yields.

• 1st year protection provided by rotation (KSU)

• Rotation to another crop for one year reduces the MCMV incidence in the next corn crop (UNL).

• Tillage reduces the risk when the residue is incorporated into the soil and decomposition results.

In Kenya, MCMV persisted in soil taken from under infected plants for at

least 49 days.

Treatmenta # Plants # Sympt. % Inf.

Control soil 23 1 4a

Infested soil 71 49 69b

Genetic Resistance/Tolerance• Last serious outbreak in

Kansas was 1988, but MCMV is still present in up to 20% of fields. Commercial hybrids are strongly resistant to potyviruses and are likely tolerant of MCMV.

• Tolerant hybrids are commercially availbableand are the best management option.

• No resistant hybrids have been developed to date; however, several display good levels of tolerance. In inoculated trials conducted in Nebraska, losses in hybrids have ranged from one to 80 percent.

Maize-infecting Potyviruses

MDMV

Myzus persicae

SCMV

Potyvirus Resistance in Maize

• Potyviruses differ in aggressiveness across resistant lines: WSMV<JGMV~SrMV<MDMV<SCMV

• Isolates of viruses can differ in aggressiveness: MDMV-Oh~SCMV-Gr<MDMV-It~SCMV-OH

• The resistance gene on chr. 6 (Mdm1/Scmv1/Wsm1) is required for resistance to MDMV, SCMV, JGMV and SrMV.

• The genes on chr. 3 (Scmv2/Wsm2) and chr. 10 (Wsm3) provide little resistance to potyviruses other than WSMV on their own.

• Wsm2 and Wsm3 increase resistance to MDMV and SCMV in the presence of Wsm1.

Multiple Virus Resistance in Oh1VI

• A single plant was selected after inoculating a Virgin Islands maize population with MCDV. The inbred line Oh1VI developed from this plant.

• Resistant to at least nine different viruses in different families that have diverse genomes and replication strategies –

– Waikiviruses (+RNA): MCDV

– Potyviruses (+RNA): MDMV, SCMV and WSMV

– Rhabdoviruses (-RNA): MFSV and MMV

– Marafivirus (+RNA): MRFV

– Tombusvirus (+RNA): MNeSV

– Emaravirus (-RNA): WMoV

• All is not rosy –– Not resistant to the fijiviruses (MRDV & MRCV), or Maize streak virus

– Twelve ft tall, wants to flower in September and is extremely susceptible to smut.

100 nm

100 nm

100 nm

100 nm

100 nm

MRFV

MFSV

MNeSVMCDV

MDMV

WMoV

cMMCDV

MMV

MFSV

MDMVSCMVWSMV

MRFV

MNeSV

QTLs

for virus

resistance

in maize

Maize LineDTFS

MCMVRank

AUDPCMCMV

RankRatingMLN

Rank

N211 26 Ad 1 105.2 5 3.83 BCD 15

KS23-6 25.6 A 2 81.1 3 2.67 A 3

DR 21.2 B 3 n.d.e n.d.

Oh1VI RIL 70332 18.8 BC 4 79.4 2 2.5 A 2

KS23-5 18.6 CD 5 71.9 1 2 A 1

Oh1VI 16.3 DE 6 104.2 7 4 BCD 18

Oh1VI RIL 70343 16.1 E 7 126.9 19 4.33 CDEF 22

Oh1VI RIL 70340 16 E 8 109.9 8 3.5 B 7

Oh1VI RIL 70294 15.9 E 9 153 35 4.83 FG 42

Oh1VI RIL 70279 15.7 E 10 117.2 13 4 BCD 17

NC358 13.2 F 11 153.6 39 5 G 47

Oh1VI RIL 70228 11.2 F 12 156 42 4.67 EFG 29

Oh28 8.2 G 13 171.8 56 5 G 59

MCMV and MLN Resistance

DTFS, days to first symptom; AUDPC, area under the disease progress curve

• MCMV was present in

all lines, as measured

by ELISA.

• Several lines derived

from Oh1VI have

significant tolerance to

MCMV.

• Resistance to U.S.

MCMV isolate and

Kenyan MCMV + SCMV

has similarities.

• MCMV present

regardless of rank.

MCMV and SCMV Resistance

Rank Entry AUDPC Entry AUDPC

1 12 13.33 43 12.13

2 14 13.33 63 13.22

3 15 13.50 64 14.38

4 16 13.33 40 14.88

5 17 13.50 54 17.22

6 23 13.33 39 18.42

7 24 13.33 42 18.63

8 26 13.33 61 17.63

9 27 13.33 53 19.17

10 29 13.33 5 18.67

Worst 53 41.60 14 40.70

• 65 EA and CIMMYT lines– SCMV-OH and MCMV-KS

– Scored for 4 wpi

• No line is in top 10 for both MCMV and SCMV

• Entry 14 among best for SCMV resistance is worst for MCMV.

• Entry 53 among best for MCMV resistance is worst for SCMV.

– Other MCMV R/SCMV S are 16 and 17.

• No correlation between SCMV and MCMV resistance in this experiment (P = 0.233).

• MCMV present regardless of rank.

Lilian Gichuru, KALRO/Borlaug Scholar

L. striatellus

MLN Control Measures

Resistant/Su

sceptible

Crop

Environment

MCMV + Potyvirus

Virus Reservioirs

Beetles/Thrips

U.S. CORNBELT EAST AFRICA

• MCMV + MDMV/WSMV

• Johnsongrass control

• Crop rotation (soil trans.)

• ‘Clean’ seed (coupled with low

vector pressure)

• Coleopteran Vectors

• Diabrotica/insect control

• Vectors less efficient?

• Resistant Tolerant Crop

• Potyvirus resistant

• MCMV tolerance available

• Environment

• Winter = 1 maize crop

• MCMV + SCMV

• Roles for luteovirus, other potyviruses?

• Common alternative hosts?

• Continuous maize

• Role of soil transmission?

• Role of seed transmission?

• Maize thrips and ?? vectors

• Efficient vector prevalent

• Mostly susceptible crop

• Potyvirus susceptible

• MCMV susceptible

• Environment

• Continuous maize

• Factors affecting thrips populations?

Some of the Many Remaining Questions….

• What is the potential rate of seed

transmission for MCMV and for SCMV?

• Does seed transmission vary by

genotype?

• What is the tolerance of seed

transmission for MCMV?

• Is MCMV presence in seed correlated

with seed transmission?

• Does MCMV tolerance affect virus titer?

• Does SCMV resistance or susceptibility

affect MCMV titer?

• Does virus resistance/tolerance affect

seed transmission?

• What is the effective maize-free period?

How is this managed?

• What are most effective rotation of seed

treatments and insecticides for

controlling MCMV and MLN in seed

production fields?

• Is there a role for beetle transmission?

• What is the persistence of MCMV in the

field? What are the best crop rotations

for managing MLN?

• What are the roles of very long (between

areas and regions), long (between fields)

and short (within field) distance

migration of thrips in MCMV incidence?

United States Department of Agriculture

Sequencea Accession

#b Lengthc Genomed nte BD8 R1 R2 R3 OH

Contig 1 KP835283 6321 BD8 416-6729 98 79 79 79 79

Contig 2 KP835284 959 Rwanda 1 4371-5328 81 99 98 98 93

Contig 10 KP835285 1835 BD8 6920-8754 99 75 75 75 73

Contig 28 KP835286 832 Rwanda 1 56-873 69 87 87 86 82

Contig

11.64KP835287 793 Rwanda 1 2914-3705 78 94 94 95 93

Contig

43.67.30.8.

31.8010.74

KP835288 1679 Rwanda 3 7165-8846 76 98 97 96 87

Contig

42.114.111.

52.101

KP835289 1560 Rwanda 1 1005-2565 73 90 90 88 86

Contig

62.16.25a.2

5b

KP835290 959 Rwanda 1 5765-6722 77 92 92 94 91

SCMV

ThailandAY629310 1144 BD8 8998-9339 98 82 82 81 80

SCMV

ThailandAY629312 1150 BD8 8192-9339 98 82 82 82 80

SCMV

KenyaJX286706 700 BD8 149-847 98 69 70 70 68

SCMV

KenyaJX286707 730 BD8 6110-6835 98 79 79 79 79

SCMV

KenyaJX286708 940 BD8 8399-9339 100 80 81 80 79

Multiplicity of SCMV

SCMV Diversity in Kenya/Uganda

• RNASeq for Kenya samples indicates two types of SCMV seq.

• RNASeq for Kenya/Uganda seq. indicates four types of SCMV seq.

• Working on relative abundance and distribution.

• SCMV sequence diversity is consistent with differential responses of antisera.

• Most variation in N-terminus of CP.

1000 2000 3000 4000 5000 6000 7000 8000 90000

P1 HC-Pro

P3 CI VPg NIa-Pro NIb CP

> 90% ID w/SCMV-Rwanda > 97% ID w/SCMV-BD8Mahuku et al. Phytopath. In press

Are there other viruses involved?

1000 2000 3000 4000 5000

74% ID to Maize yellow dwarf virus

L. striatellus

MLN is an occasional and local

problem in the mainland U.S.

Control Measures include:

• Winter

• Living with the disease

• Crop rotation

• Virus resistance/tolerance

• Weed control, especially

Johnsongrass

• No complacency• Jardine http://www.plantpath.ksu.edu/pages/extension/factsheets• Uyemoto 1983. Plant Dis. 67: 7- 10.

• Stack (UNL) https://pdc.unl.edu/agriculturecrops/corn/maizechloroticmottlevirus

• Nelson et al. http://www.ctahr.hawaii.edu/oc/freepubs/pdf/PD-79.pdf

MCMV Control in the U.S.

MLN is a more chronic problem in Hawaii.

Control measures include:

• Apply insecticide sprays, weekly if necessary, to control thrips vectors.

• Control alternate weed hosts, especially grassy weeds, to reduce MCMV reservoir and corn thrips populations

• Scout fields regularly and remove any symptomatic plants.

• Keep unnecessary people and machines out of the field to reduce mechanical transmission and spread of MCMV.

•