Paecilomyces Lilacinus 1000 P=0.002 Melocon in Citrus

Nematodes… The phylum Nematoda (Nemata) is comprised of aquatic roundworms that occupy virtually all habitats on earth. Nematodes are the most numerous metazoans on earth and the phylum is one of the most species-diverse. It has been estimated that only 3% of a half million species have been described. Nematodes are adapted to numerous life strategies with major trophic groups consisting of primary (plant parasites) and secondary consumers (bacterivores, fungivores, omnivores, predators, and animal parasites). They play a critical role in the fertility of agricultural soils as major contributors to nutrient decomposition in soil food webs, and their ubiquity makes them ideal organisms to study as biological indicators of soil processes. In contrast to the beneficial roles played by nematodes, all plant and animal species have nematode parasites. Parasitism of crop plants by some nematode species is of little economic significance, whereas others are among the most serious agricultural pests. 1 Nematodes…. Things you should know  Roundworms  Aquatic  Ubiquitous  Anhydrobiotic ability (trehalose, glycerol)  Most trophic groups represented (used as bioindicators)  Importance of Caenorhabditis elegans as a biological model (900 cells, genome sequenced, short life cycle). Nervous Digestive Reproductive Excretory Muscle Hydrostactic skeleton (osmoregulate)

2 Predators

Bacterivores

Free living nematode feeding habits Fungivores

(trophic groups) 3 Mosquito vectored diseases Animal Elephantiasis parasites Intestinal parasites

Pinworms in children

Hookworms Dog heartworm

Vectored by blackflies

Guinea worm Trichinosis in pigs and game.

4 Insect parasites (Actually, bacterial feeders)

5 Plant parasites

6 styletstylet metacorpus Median(pump) eggs bulb vulva glands for extraoral digestion and feeding site establishment ovary

intestine anus

7 Some nematodes have coevolved with their host plants to a very high degree. They exhibit remarkably complex feeding behaviors and responses to chemical cues. Nematode head Feeding tube (ft = molecular filter) in cytoplasm, typical of several genera of sedentary endoparasites. Notice how the plasmalemma (Pb) is invaginated, rather than punctured by the nematode stylet (S).

8 …..Whereas other nematodes exibit less specialized feeding behavior. These Radopholus similis simply burrow through the root cortex, feeding on cells that are not destroyed by previous nematode feeding or by fungi and bacteria that invade the tunnels created by the nematodes.

9 Nematodes can be classified by their feeding habits. Ectoparasites migratory (sting) sedentary Endoparasites migratory (burrowing and lesion) sedentary (root-knot) Semi-endoparasites migratory sedentary (citrus) Host plant resistance is more commonly encountered for the sedentary endoparasites – why do you think this is?

10 What you should know about nematode IPM in citrus Citrus Nematode (Tylenchulus semipenetrans) Sedentary semi-endoparasite Coevolved with citrus (least virulent, specialized feeding cells, several biotypes, narrow host range beyond rutaceae, population density regulates importance) Burrowing Nematode (Radopholus similis) Migratory endoparasite Wide host range Problem only on Florida ridge (understanding why important for management) Phylogeny recently revised (information that could be important for management) Coffeae Lesion Nematode (Pratylenchus coffeae) Biology like burrowing nematode Species complex (illustrates importance of taxonomy to IPM) Rare, but most virulent species Sting nematode (Belonolaimus longicaudatus) Young tree pathogen (Why?) Prefers moist soil Obvious root symptoms 11 Break !

12 The Natural History Guiding Nematode IPM in Orchards Larry Duncan University of Florida Citrus Research and Education Center The Natural History Guiding Nematode IPM in Orchards

Integrated Pest Management  Sanitation  Host plant resistance/tolerance  Cultural practices  Chemical and biological pesticides The Natural History Guiding Nematode IPM in Orchards Phylogenetics Taxonomy Populations  Biology/Ecology  Spatial Patterns  Temporal Patterns The Natural History Guiding Nematode IPM in Orchards

Questions that can only be addressed by knowing what happens in nature.

Controlled laboratory experiments are inadequate. Nematode-induced tree delines

Sedentary Citrus nematode, Tylenchulus semipenetrans Endoparasite (Slow decline) Burrowing nematode, Radopholus similis (Spreading decline) Migratory Endoparasite Coffee lesion nematode, Pratylenchus coffeae (Citrus slump)

Migratory Sting nematode, Belonolaimus longicaudatus Ectoparasite Dagger nematode, Xiphinema vulgare Nematode-induced tree delines

Migratory Sting nematode, Belonolaimus longicaudatus Ectoparasite Dagger nematode, Xiphinema vulgare Tylenchulus semipenetrans

 Co-evolved with citrus

 Remarkably well-adapted parasite

 Economic importance depends on population density which varies with habitat Question - Why are citrus nematodes most abundant in dry soil conditions? Citrus nematode is not well-adapted to survive desiccation (Van Gundy et al., 1964: Tsai and Van Gundy, 1989), but:

Anhydrobiosis requires behavioral and biochemical changes.

Coiling and aggregating reduce the rate of water loss.

This provides time for conversion of glycogen or lipid to the non-reactive sugar trehalose. Trehalose replaces bound water on membranes so that proteins do not become denatured by coming into contact and reacting with one another through sulfide bonding. Question - Why are citrus nematodes most abundant in dry soil conditions?

Citrus nematode is not well-adapted to survive desiccation (Van Gundy et al., 1964: Tsai and Van Gundy, 1989), but: ______Climate Population Population density dynamics (crop loss) ______

Humid Marked Lower Tropics seasonality

Mediterranean Seasonality less Higher Climates apparent Citrus nematode and Phytophthora nicotianae are regulated by seasonal carbon flux in fibrous roots. This was subsequently validated experimentally by removing developing citrus fruit to increase carbohydrate levels in roots.

Phytopathology 1993, 83:573-581 Linear correlation coefficients between average monthly population estimates and selected environmental variables. ______T. semipenetrans P. nicotianae J2 Fem CFU/g ng protein/ Variable per sample soil g root ______Soil temperature -0.18 -0.35 0.06 0.51 ** Soil moisture -0.59 ** -0.54 ** -0.17 0.25 Root mass density 0.71 ** 0.77 ** 0.07 0.26 Nonstructural carbohydrate 0.53 ** 0.58 ** -0.36 0.10 Starch 0.48 ** 0.56 ** -0.49 ** -0.21 Ketone sugars 0.34 0.11 0.30 0.34 Reducing sugars 0.61 ** 0.48 * -0.17 0.03 Free glucose 0.11 0.15 0.23 -0.30 Lignin -0.54 ** -0.55 ** 0.15 0.09 Bioassay 0.35 0.21 -0.20 0.44 ______Best-fit predictive variables for population density from stepwise multiple regression

Variable R2

T. semipenetrans Soil moisture (-) 0.62 Starch 0.73

P. nicotianae (%protein) Soil temperature 0.51 Ketone sugars 0.63 Soil moisture 0.76

P. nicotianae (cfu) Ketone sugar 0.75 Root mass density 0.86

Effect of hydraulic lift on population dynamics of Tylenchulus semipenetrans

The soil moisture and nematodes were measured in just the top pot.

Question - Why does soil salinity favor citrus nematode in the field but not in the laboratory? ______Direct relationship between salinity and population density in field plots. Machmer (1958) Higher nematode population density in saline soils of Sundays River Valley. Cohn (1976) But Greenhouse studies revealed no or negative effect of salinity on T. semipenetrans. Kirkpatrick and Van Gundy (1966) Experimental protocol (P. Mashela) ______

Grow plants in non-saline and saline conditions

After 6 weeks, flush salts from some pots to create the following treatments: No salinity Continuous salinity Discontinuous salinity

Add citrus nematodes to pots 1200 500 400 1000 300 SoilEC 200 100 800 C REC N40 PCD PCW Salinity as measure by 600 soil EC varies seasonally, SoilEC 400 depending on whether 200 precipitation is in the 0 form of irrigation water 6.5 CREC 8 6.0 N40 or rainfall. Thus, the

SoilpH 5.5 5.0 PCD C REC N40 PCD PCW PCW plant and nematode 7 experiences alternating SoilpH stressful and non- 6 stressful conditions.

5 M A A M J J A A S O N D J F M A M J J A S O N D Month 500 No salinity Salinity Population 400 Intermitten salinity growth increased on 300 salt stressed 200 plants only after salinity 100

Numbereggs femalesand was removed – 0 This is what Females Eggs happens in rainy season.

Summary ______

Salinity increases population density of citrus nematodes Citrus nematodes cause: Leaves Increased Cl Increased Na Decreased K Roots Decreased Cl Decreased Na Decreased K ______Nematode-induced tree delines

Migratory Sting nematode, Belonolaimus longicaudatus Ectoparasite Dagger nematode, Xiphinema vulgare

Healthy Sting nematode damaged Question - Why are sting nematodes more numerous on small than large trees ______Damage functions for sting nematodes on citrus. Trees were 5 years old. These kinds of relationships are expected for field crops that are harvested soon after planting. For these perennial plants they do not seem reasonable. Why do the large trees with many roots support fewer nematodes than small trees with few roots? Soil texture and depth is the same for all trees. Log sting nematode/100 cm3 soil Growers apply irrigation when needed by larger trees, so soil on small trees remains wet rather than experiencing wet and dry cycles. Why is Sting nematode only recently a problem?

Cultural practices changed. Mowing and herbicides replaced disking for weed control.

Sting nematode has a wide host range and numbers increased on the vegetation in the row middles.

When groves were replanted after freezes in 1980s, trees planted in patches of high sting density were stunted, whereas trees planted in soil with low density grew enough to initiate the wet- dry soil cycles that force the nematode below the zone of highest root density near the soil surface. TH CREC

IT PL Note that DNA evidence suggests that, like P. coffeae, B. longicaudatus also appears to be a species complex. Mating studies can help to confirm this.

Topotype population of B. longicaudatus True B. longicaudatus is on East Coast. Other genotypes appear to be geographically segregated to some extent. The southeastern US is the primary location for sting nematodes and the largest Flatwoods Shallow soil numbers of species will Variable texture occur nearest the center of origin. Nematode-induced tree delines

Migratory Sting nematode, Belonolaimus longicaudatus Ectoparasite Dagger nematode, Xiphinema vulgare Citrus slump Citrus slump

Note similarity to symptoms of burrowing nematode. However, lesion nematode damage is not restricted to the deeper roots, so it is a more serious pathogen. Fortunately its distribution is very limited and the nursery certification program helps prevent its spread.

No resistance, so most damaging species if present. Pratylenchus coffeae is a regulated species in Florida citrus nurseries. Nursery site certification surveys frequently detected P. coffeae on native plants and ornamental Musa and Ficus. P. coffeae detection frequency at proposed nursery sites was much higher than in citrus orchards. P. coffeae is a pest of citrus in some countries, but not others.

P. coffeae likely species complex. Pratylenchus loosi from pasture grasses in Central Florida. Nematologica 42:159-172. Taxonomic survey 39 Pratylenchus coffeae isolates were collected from coffee, citrus, yam, Musa, etc. Isolates were gotten from S.E. Asia, Middle East, Africa, Caribbean, S. America, Central America and Florida. Identification Presence or absence of males Number lip annules Tail shape Morphometric values Body diam. at vulva, mid body, metacorpus. Body length Esophagus length. Post uterine branch length. Tail length Ratios a,b, b’, c, c’ and Percentages V Lip patterns (observed with SEM) are very useful to characterize root lesion nematodes and were first used to discriminate P. gutierrezi from P. coffeae. In our survey, just 3 isolates from coffee in Central America (including topotype P. gutierrezi) and 1 isolate from Aster in Florida had divided faces. Faces.faces. -3 -2 -1 0 1 2 3 4

A. K5 K2 C7 N1C N1 C1 K4 N3 2 A1 C2 K3 A1C B4 T N2 The morphometric A1C A1 M1 Y1 characters considered in 0 K6 N2C B3 K1 total did not M3 C3 C3C M3C Y3 B2 M2 -2 discriminate distinct B1 C6 groups of isolates as well Y2 C3 -4 C5 C4

VII K3 as a few non-allometric 2 B.

K4 A1C K2C characters (lip III A1C A1 B3

Principal component 2 1 K1 Y2 A1 morphology, stylet M3 Y3 B1C I C5 M1C II C4 M3C C3 K6 M2C C3C Y1 B2C length, V, a). 0 K8 K9 K10 C6 K7 C3 B4 TC IV

C1 -1 V K5 C2 C7

N3 N2C N1C -2 VI N1

-2 -1 0 1 2 Principal component 1 DNA (d2/d3) was sequenced by Kelly Thomas, Linda Friese and Krystline Morris for phylogenetic analysis. Note congruence (unusual) between the molecular and morphological relatedness of these isolates. Taxonomic characterization was aided by topotype material lacking in earlier reports. K6 = P. coffeae K3 = P. gutierrezi T = P. loosi

C4 (Florida citrus) is P. coffeae Subsequently,

C1 = P. jaehni (2001). N1 = P. floridensis (2010). N2 = P. parafloridensis (2010) B1 = P. speijeri (2012)

C2, K1 and K2 remain undescribed. Morphological characters of a topotype (k6) population of Pratylenchus coffeae from Indonesia as proposed by Inserra et al., 2001. Subsequent validatin by Nguyen, 2010 and De Luca et al., 2012. P. floridensis P. parafloridensis

P. speijeri

P. coffeae P. jaehni Nematode-induced tree delines

Migratory Sting nematode, Belonolaimus longicaudatus Ectoparasite Dagger nematode, Xiphinema vulgare Spreading decline caused by the citrus race of Radopholus similis the burrowing nematode Spreading decline caused by Radopholus similis the burrowing nematode Sanitation

Commercial nurseries must be certified free of citrus, burrowing and coffee lesion nematodes

Initiated in response to spreading decline

No other industry does this

Management of nematodes in the nursery only disguises the problem and spreads the nematodes into the orchards % Orchards infested by T. semipenetrans

Morocco 100 Texas 90 Arizona 90 Israel 90 Australia 90 Spain 90 Egypt 90 South Africa 90 Taiwan 90 Philippines 90

California 50 Florida (1977) 50

Florida (1996) 15 Florida Citrus Rootstock Certification Program 1994-1995

32.5 million dollars additional on-tree values by restricting the spread of T. semipenetrans

Annual cost of program was $70,000

464 : 1 (benefit : cost)

- Paul Lehman Florida Division of Plant Industry Florida Citrus Rootstock Certification Program 1994-1995

32.5 million dollars additional on-tree values by restricting the spread of T. semipenetrans

Annual cost of program was $70,000

464 : 1 (benefit : cost)

- Paul Lehman Florida Division of Plant Industry

Similar savings from managing Burrowing nematode Burrowing nematode is a problem for citrus only on the deep sands

Citrus is grown in of the central ridge. two ecoregions

 Central ridge Not a problem in the  Flatwoods shallow soils of the flatwoods ecoregion 95 pounds of fibrous roots on 20-year-old healthy tree vs 35 pounds of roots on tree infected by burrowing nematode. Prior to widespread irrigation, trees required deep roots for access to water during the dry winter-spring season in Florida Cultural practices to manage burrowing nematode Certified trees Resistant rootstocks Kuharski Carrizo Milam lemon Ridge pineapple Protect shallow roots Supplemental irrigation Fertigation No disking for weed control Nematode management tactics … …in order of importance ______

Sanitation

Commercial nurseries must be certified free of citrus, burrowing and coffee lesion nematodes

Initiated in response to spreading decline

No other industry does this

70 % orchards infested by T. semipenetrans

Morocco 100 Texas 90 Arizona 90 Israel 90 Australia 90 Spain 90 Egypt 90 South Africa 90 Taiwan 90 Philippines 90

California 50 Florida (1977) 50

Florida (1996) 15 71 Florida Citrus Rootstock Certification Program 1994-1995

32.5 million dollars additional on-tree values by restricting the spread of T. semipenetrans

Annual cost of program was $70,000

464 : 1 (benefit : cost)

- Paul Lehman Florida Division of Plant Industry

72 Florida Citrus Rootstock Certification Program 1994-1995

32.5 million dollars additional on-tree values by restricting the spread of T. semipenetrans

Annual cost of program was $70,000

464 : 1 (benefit : cost)

- Paul Lehman Florida Division of Plant Industry

Similar savings from managing Burrowing nematode 73 Nematode management tactics … …in order of importance ______

Sanitation

Rootstock resistance

Cultural practices

Biological control

Chemical control

74 Rootstock resistance

Citrus nematode Swingle citrumelo (citrus nematode)

Burrowing nematode Milam lemon (burrowing nematode) Ridge pineapple (burrowing nematode) Kuharski Carrizo (burrowing nematode)

Resistance-breaking biotypes

Resetting vs Replanting (which makes sense?) 75 Sunn Hemp and perennial peanut are non-host plants for sting nematode. Should we grow them in row-middles where the nematode is a problem? Burrowing nematode Lesion nematode

Flattened lips Ventral overlap of glands

Dorsal overlap of glands Rounded lips Which 2 are cousins?

Globodera Cyst nematode

Radopholus Burrowing nematode

Pratylenchus Lesion nematode Cyst nematodes

Burrowing nematode

Lesion nematodes

Subbotin et al., 2006, Nematology 8: 455-474 Transformed potato in 2002 to express a cystatin that disrupts digestion of certain proteins and also a peptide that interferes with sensory function of cyst nematodes.

In 2012 transformed banana with these genes to confer resistance to burrowing nematode. Heavy use of nematicides currently required to grow banana. There is resistance to releasing transgenic plants for crop production.

Huanglongbing is such a devasting disease in Florida’s citrus industry that transgenic crops have been approved for commercial production.

This may provide a possibility to engineer resistance to the burrowing nematode in citrus rootstocks.

Especially if the trans-gene expression is limited to the rootstock. AtMDK-20 (root cap promoter)

Tub-1 promoter

Lilley et al. 2004 & 2011. Plant Biotechnology Journal 2: 3-12 Plant Biotechnology Journal 9:151–161 RB-7 promoter Nematode management tactics … …in order of importance ______

Sanitation

Rootstock resistance

Cultural practices

Biological control

Chemical control

83 Nematode management tactics … …in order of importance ______

Sanitation

Rootstock resistance

Cultural practices

Biological control

Chemical control

84 Cultural practices

Limiting factors? (all nematodes) Poor drainage Drought Salinity Nutritional deficiencies / toxicities Phytophthora nicotianae

Shallow root function (burrowing nematode) Supplemental irrigation Fertigation No disking (Protect shallow roots!) 85 Nematode management tactics … …in order of importance ______

Sanitation

Rootstock resistance

Cultural practices

Biological control (not yet)

Chemical control

86 Biological control (not yet)

87 Works in lab conditions. Challenge is making it work in field. So far not working in field for Paecilomyces lilacinus 1000 P=0.002 MeloCon in citrus. Not yet tested Pasteuria in 750 field. 500 soil

3 250 0 2nd Biological 1000 P=0.007

per 100 cm 100 per 750 500 Number citrus nematodes Number 250 0 Treated Control

Biological control (not yet)

88 But there is surprisingly little known about 12 Untreated Paecilomyces lilacinus subterranean biological control. Paecilomyces 10 P < 0.05 lilacinus not considered an entomopathogen, but 8 note our results from field trial last year measuring

6 nematodes and insects. No effect on nematodes,

cumulative 4 significant effect on weevils ….?

Mean weevils per plot per weevils Mean 2

0 50 100 150 200 250 300 350 Julian Days

Biological control (not yet)

89 Nematode management tactics … …in order of importance ______

Sanitation

Rootstock resistance

Cultural practices

Biological control

Chemical control

90 Chemical control

Monitoring Multiple applications Accelerated microbial degradation

91 Taking a soil sample forSampling monitoring with plant a parasiticsoil probenematodes.

92 You may hear about this.

93 Fewer nematodes in shallow roots, but easier to collect large quantity of surface roots with a shovel

94 Samples of surface roots collected with a shovel detected nematode infected trees more efficiently than samples collected by jeep mounted augers

95 Chemical control

Monitoring Multiple applications Accelerated microbial degradation

96 With recent deregistration of Temik, Vydate is the only nematicide registered for use on citrus in Florida. Vydate is systemic with basipetal movement in the tree, which makes it effective against burrowing nematode.

Protect your groundwater! Treat in dry season Avoid treating near wells Only treat when warranted by sampling

97 1, 3-dichloropropene (Telone); enzyme inhibitor; mamal LD50 519 mg/kg; fumigant nematicide.

Aldicarb (Temik – deregistered 2011); carbamate; reversable acetylcholinesterase inhibitor; mamal LD50 1 mg/kg.

Oxamyl (Vydate); organophosphate; irreversable acetylcholinesterase inhibitor; mamal LD50 3 mg/kg.

Fluopyram (Velum Prime); succinate dehydrogenase inhibitor; mamal LD50 >2000 mg/kg; Fungicide, nematicide.

Spirotetramat (Movento); lipid biosynthesis inhibitor; mamal LD50 >2000 mg/kg; insecticide, nematicide.