Morphological and Molecular Characterization of Meloidogyne mayaguensis Isolates from Florida

The discovery of Meloidogyne mayaguensis is confirmed in Florida; this is the first report for the continental United States. Meloidogyne mayaguensis is a virulent species that can reproduce on host cultivars bred for nematode resistance. The perineal patterns of M. mayaguensis isolates from Florida show morphological variability and often are similar to M. incognita. Useful morphological characters for the separation of M. mayaguensis from M. incognita from Florida are the male stylet length values (smaller for M. mayaguensis than M. incognita) and J2 tail length values (greater for M. mayaguensis than M. incognita). Meloidogyne mayaguensis values for these characters overlap with those of M. arenaria and M. javanica from Florida. Enzyme analyses of Florida M. mayaguensis isolates show two major bands (VS1-S1 phenotype) of esterase activity, and one strong malate dehydrogenase band (Rm 1.4) plus two additional weak bands that migrated close together. Their detection requires larger amounts of homogenates from several females. Amplification of two separate regions of mitochondrial DNA resulted in products of a unique size. PCR primers embedded in the COII and 16S genes produced a product size of 705 bp, and amplification of the 63-bp repeat region resulted in a single product of 322 bp. Nucleotide sequence comparison of these mitochondrial products together with sequence from 18S rDNA and ITS1 from the nuclear genome were nearly identical with the corresponding regions from a M. mayaguensis isolate from Mayaguez, Puerto Rico, the type locality of the species. Meloidogyne mayaguensis reproduced on cotton, pepper, tobacco, and watermelon but not on peanut. Preliminary results indicate the M. mayaguensis isolates from Florida can reproduce on tomato containing the Mi gene. Molecular techniques for the identification of M. mayaguensis will be particularly useful in cases of M. mayaguensis populations mixed with M. arenaria, M. incognita, and M. javanica, which are the most economically important root-knot nematode species in Florida, and especially when low (<25) numbers of specimens of these species are recovered from the soil.     

Population Development of Pasteuria penetrans on Meloidogyne arenaria

A microplot study on the influence of cropping sequences with peanut in summer and bare fallowed or cover crops of rye or vetch in winter on the population development of Pasteuria penetrans was initiated in the spring of 1987. The number of spores of P. penetrans attached per second-stage juvenile of Meloidogyne arenaria race 1 increased from 0.11 in the fall of 1987 to 7.6, 8.6, and 3.6 in the fall of 1989 in the rye, vetch, and fallowed plots, respectively. Higher (P ≤ 0.05) levels of P. penetrans occurred in the rye and vetch plots than in fallowed plots. No influence of P. penetrans on peanut, rye, or vetch yield was observed in 1987 and 1988, but in 1989 peanut yield was 64% higher (P ≤ 0.05) in plots infested with P. penetrans than in plots without P. penetrans. Numbers of M. arenaria in plots without P. penetrans were influenced by the cropping sequences in the spring of 1988 and 1989 but not in the fall following the peanut crop. In the spring the plots with rye had the lowest nematode numbers in either year (P ≤ 0.05). Nematode numbers were lower (P ≤ 0.05) in plots with P. penetrans than in plots without P. penetrans in the spring of 1989 (vetch) and the fall of 1989 (rye, vetch, and fallowed).     

Evaluation of Morphological Variability in Meloidogyne arenaria

Seven populations, representing cytological race A (triploid, 3n = 51-56) and the two host races (infective and noninfective on peanut) of Meloidogyne arenaria were studied with light microscopy (LM) and scanning electron microscopy (SEM). Characteristics of root-knot nematodes, recently recommended as useful taxonomic traits, were reexamined among these populations, and their variability both within and between populations was ascertained. We found that stylet morphology of females and head and stylet morphologies of males and second-stage juveniles were the most reliable characters for identification. The two host races of M. arenaria could not be distinguished morphologically. Two of the populations could be separated consistently from the remainder but were not sufficiently divergent to be considered new species. These two variant populations were similar; neither produced males in culture, and they differed from the typical populations in female perineal patterns (LM) as well as in cephalic structure (SEM) and tail shape (LM) of second-stage juveniles. In morphometric studies, most characters of the variant populations differed significantly from those of the typical M. arenaria.     

Resistance of maize to Meloidogyne arenaria and Meloidogyne javanica

Summary A diallel cross of eight maize, Zea mays L., inbred lines was analyzed for reaction to two species of root-knot nematodes, Meloidogyne arenaria (Neal) Chitwood and M. javanica (Treub) Chitwood. Egg production following inoculation of F₁ hybrid seedlings with nematode eggs was determined in a greenhouse experiment. Data were analyzed using Griffing's Method 4, Model I. General combining ability was a significant source of variation in egg production of both M. arenaria and M. javanica; specific combining ability was not a significant source of variation for either. The correlation between egg production of the two nematode species on the 28 F₁ hybrids was highly significant. Hybrids with Mp313 or SC213 as one parent were the most resistant to both species. This indicates that germ plasm is available for developing inbred lines and hybrids with resistance to both M. arenaria race 2 and M. javanica.This article is a contribution of the Crop Science Research Laboratory, U.S. Department of Agriculture, Agricultural Research Service, in cooperation with the Mississippi Agricultural and Forestry Experiment Station, Journal No. J-7481.     

Population Dynamics of Meloidogyne arenaria and Pasteuria penetrans in a Long-Term Crop Rotation Study

The endospore-forming bacterium Pasteuria penetrans is an obligate parasite of root-knot nematodes (Meloidogyne spp.). The primary objective of this study was to determine the effect of crop sequence on abundance of P. penetrans. The experiment was conducted from 2000 to 2008 at a field site naturally infested with both the bacterium and its host Meloidogyne arenaria and included the following crop sequences: continuous peanut (Arachis hypogaea) (P-P-P) and peanut rotated with either 2 years of corn (Zea mays) (C-C-P), 1 year each of cotton (Gossypium hirsutum) and corn (Ct-C-P), or 1 year each of corn and a vegetable (V-C-P). The vegetable was a double crop of sweet corn and eggplant (Solanum melongena). A bioassay with second-stage juveniles (J2) of M. arenaria from a greenhouse (GH) population was used to estimate endospore abundance under the different crop sequences. A greater numerical increase in endospore densities was expected in the P-P-P and V-C-P sequences than in the other sequences because both peanut and eggplant are good hosts for M. arenaria. However, endospore densities, as determined by bioassay, did not substantially increase in any of the sequences during the 9-year experiment. To determine whether the nematode population had developed resistance to the resident P. penetrans, five single egg-mass (SEM) lines from the field population of M. arenaria were tested alongside the GH population for acquisition of endospores from the field soil. Four of the five SEM lines acquired 9 to 14 spores/J2 whereas the GH population and one of the SEM lines acquired 3.5 and 1.8 spores/J2, respectively. Endospore densities estimated with the four receptive SEM lines were highest in the P-P-P plots (14-20 spores/J2), intermediate in the V-C-P plots (6-7 spores/J2), and lowest in the Ct-C-P plots (< 1 spore/J2). These results indicate that the field population of M. arenaria is heterogeneous for attachment of P. penetrans endospores. Moreover, spore densities increased under intensive cropping of hosts for M. arenaria, but the GH population of the nematode was not receptive to spore attachment. However, previously, the GH population was very receptive to spore acquisition from this field site. One explanation for this inconsistency is that the M. arenaria population in the field became resistant to the dominant subpopulation of P. penetrans that had been present, and this led to the selection of a different subpopulation of the bacterium that is incompatible with the GH population.     

Morphological Comparison of Seven Hypotriploid Populations of Meloidogyne arenaria with the Typical Triploid Populations

A morphological comparison of seven hypotriploid populations of Meloidogyne arenaria was made to clarify their taxonomic status, using light and scanning electron microscopy. All populations differed from each other and from the typical triploid M. arenaria by certain features. Differences were not regarded as sufficient to justify recognition of the variants as distinct species. Morphological divergence of populations from the typical M. arenaria was gradual. The most useful characters were stylet and head morphology of males and stylet morphology of females. Perineal patterns and cephalic, stylet, and tail morphologies of second-stage juveniles were of little taxonomic value. Host races 1 and 2 could not be distinguished morphologically. Populations E445 and E551 with the atypical esterase phenotypes M3-F1 and S1-M1, respectively, were morphologically more similar to the typical M. arenaria than populations E255 and E467, which have the most common A2 esterase phenotype of M. arenaria.     

Morphological and Molecular Evaluation of a Meloidogyne hapla Population Damaging Coffee (Coffea arabica) in Maui,Hawaii

An unusual population of Meloidogyne hapla, earlier thought to be an undescribed species, was found causing large galls, without adventitious roots, and substantial damage to coffee in Maui, Hawaii. Only in Brazil had similar damage to coffee been reported by this species. Unlike M. exigua from South and Central America, this population reproduced well on coffee cv. Mokka and M. incognita-susceptible tomato but poorly on tomato with the Mi resistance gene. Characterization included SEM images, esterase isozymes, and five DNA sequences: i) the D3 segment of the large subunit (LSU-D3 or 28S) rDNA, ii) internal transcribed spacer (ITS-1) rDNA, iii) intergenic spacer (IGS) rDNA, iv) the mitochondrial interval from cytochrome oxidase (CO II) to 16S mtDNA, and v) the nuclear gene Hsp90. Sequences for ITS-1, IGS, and COII were similar to other M. hapla populations, but within species ITS-1 variability was not less than among species. One LSU-D3 haplotype was similar to a previously analyzed population with two minor haplotypes. Hsp90 exhibited some variation between Maryland and Hawaiian populations distinct from other species. Females were narrow with wide vulval slits, large interphasmidial distances, and more posterior excretory pores; 20% of perineal patterns had atypical perivulval lines. Males had a low b ratio (<12 µm). Juveniles had a short distance between stylet and dorsal gland orifice. Juvenile body length was short (<355 µm) and was different between summer and winter populations.     

Effects of Peanut-Tobacco Rotations on Population Dynamics of Meloidogyne arenaria in Mixed Race Populations

A 3-year microplot study was initiated to characterize the population dynamics, reproduction potential, and survivorship of single or mixed populations of Meloidogyne arenaria race 1 (Ma1) and race 2 (Ma2), as affected by crop rotations of peanut ''Florigiant'' and M. incognita races 1 and 3-resistant ''McNair 373'' and susceptible ''Coker 371-Gold'' tobacco. Infection, reproduction, and root damage by Ma2 on peanut and by Ma1 on resistant tobacco were limited in the first year. Infection, reproduction, and root-damage potentials on susceptible tobacco were similar for Ma1 and Ma2. In the mixed (1:1) population, Ma1 was dominant on peanut and Ma2 was dominant on both tobacco cultivars. Crop rotation affected the population dynamics of different nematode races. For years 2 and 3, the low numbers of Ma1 and Ma2 from a previous-year poor host increased rapidly on suitable hosts. Ma1 had greater reproduction factors ([RF] = population density at harvest/population density at preplandng) than did Ma2 and Ma1 + Ma2 in second-year peanut plots following first-year resistant tobacco, and in third-year peanut plots following second-year tobacco. In mixed infestations, Ma1 predominated over Ma2 in previous-year peanut plots, whereas Ma2 predominated over Ma1 in previous-year tobacco plots. Moderate damage on resistant tobacco was induced by Ma1 in the second year. In the third year, moderate damage on peanut was associated with ''Ma2'' from previous-year peanut plots. The resistant tobacco supported sufficient reproduction of Ma1 over 2 years to effect moderate damage and yield suppression to peanut in year 3.     

Cloning and characterization of an extremely conserved satellite DNA family from the root-knot nematode Meloidogyne arenaria.

A new satellite DNA family, named pMaE, has been cloned from the genome of the phytoparasitic nematode, Meloidogyne arenaria (Nematoda: Tylenchida). It is represented as tandemly repeated sequences with a monomeric unit of 172 bp. The monomers are present at approximately 15700 copies per haploid genome, and represent about 5.3% of the total genomic DNA. Twenty-seven independent monomers have been cloned and sequenced. The deduced consensus sequence is 70.9% A + T rich, with frequent stretches of A and (or) T. Several direct or inverted sub-repeats are present in the sequence, which may allow the formation of a dyad structure, suggesting some potential role of this repetitive sequence in heterochromatin condensation. The monomers are very homogeneous in sequence, showing on average 1.8% divergence from their consensus sequence. Moreover, Southern blot experiments and sequence analysis of homologous monomers from the genome of geographically distinct M. arenaria populations have shown that this satellite DNA is uniformly distributed and highly conserved within the species. Therefore, it is hypothesized that this unusually low level of variability, either within the genome of a given population or between populations, could be achieved as the result of some highly effective homogenization mechanism acting upon the nematode genome.     

Interactions of arbuscular mycorrhizae,Meloidogyne arenaria,and phosphorus fertilization on peanut

 The individual and combined effects of two arbuscular mycorrhizal fungi (AMF), Meloidogyne arenaria, and phosphorus (P) fertilization, (0, 25, 75, and 125 μg/g soil) on peanut plant growth and pod yield were determined in greenhouse studies. Best growth and yield usually occurred at 75 or 125 μg P regardless of inoculation treatment. Peanut growth and yield were generally stimulated by AMF development, and growth alone was suppressed by M. arenaria at 0 and 25 μg P. In challenge inoculations, VAM increased peanut plant tolerance to the nematode and offset the growth reductions caused by M. arenaria at the two lower P levels. However, VAM and added P increased galling and M. arenaria egg production/g root, thereby increasing peanut susceptibility to nematode attack. M. arenaria had only a minimal effect on root colonization by AMF and sporulation by the fungi. Accepted: 9 June 1995     

Morphological and Molecular Characterization of Discocriconemella inarata, an Endemic Nematode from North American Native Tallgrass Prairies

Discocriconemella inarata, a plant parasitic nematode species originally discovered in a virgin tallgrass prairie in northwest Iowa, was re-examined by molecular and morphological analyses of topotype material. This species has never been recorded in cultivated fields and could potentially serve as an indicator for high quality prairie habitats. DNA sequence from a conserved 3' portion of the 18S ribosomal gene exhibited an identical match between D. inarata topotype specimens and topotype specimens of Mesocriconema xenoplax from Fresno, California. Higher resolution sequence analyses using the internal transcribed spacer 1 (ITS1) and a portion of the mitochondrial gene cytochrome b (cytb) allowed discrimination of D. inarata apart from M. xenoplax. This pair of species formed a well-supported clade with other Mesocriconema species exclusive of tropical Discocriconemella species. Scanning electron microscopy confirmed the absence of submedian lobes on D. inarata, suggesting a secondary loss of this defining morphological characteristic for Mesocriconema. Observations and measurements of D. inarata juveniles were added for the first time. Surveys of other prairies within the Great Plains expanded the known distribution of this species.     

Serological Relationship of Meloidogyne incognita and M. arenaria

Eight to ten precipitin bands were formed in a double immunodiffusion system comparing antigens of adult females of Meloidogyne incognita and M. arenaria. Most of the precipitin bands, based on band position and coalescence, were common to both species. Antiserum specific for M. incognita was prepared by cross absorption. Two populations of M. incognita were serologically identical, whereas two populations of M. arenaria differed slightly with respect to one weak precipitin band.     

Effects of Tropical Rotation Crops on Meloidogyne arenaria Population Densities and Vegetable Yields in Microplots

The effects of 12 summer crop rotation treatments on population densities of Meloidogyne arenaria race 1 and on yields of subsequent spring vegetable crops were determined in microplots. The crop sequence was: (i) rotation crops during summer 1991 ; (ii) cover crop of rye (Secale cereale) during winter 1991-92; (iii) squash (Cucurbita pepo) during spring 1992; (iv) rotation crops during summer 1992; (v) rye during winter 1992-93; (vi) eggplant (Solanum melongena) during spring 1993. The 12 rotation treatments were castor (Ricinus communis), cotton (Gossypium hirsutum), velvetbean (Mucuna deeringiana), crotalaria (Crotalaria spectabilis), fallow, hairy indigo (Indigofera hirsuta), American jointvetch (Aeschynomene americana), sorghum-sudangrass (Sorghum bicolor x S. sudanense), soybean (Glycine max), horsebean (Canavalia ensiformis), sesame (Sesamum indicum), and peanut (Arachis hypogaea). Compared to peanut, the first eight rotation treatments resulted in lower (P ≤ 0.05) numbers of M. arenaria juveniles on most sampling dates. Soybean, horsebean, and sesame rotations were less effective in suppressing nematodes. Yield of squash was greater (P ≤ 0.05) following castor, cotton, velvetbean, and crotalaria than following peanut. Compared to the peanut rotation, yield of eggplant was enhanced (P ≤ 0.10) following castor, crotalaria, hairy indigo, American jointvetch, and sorghum-sudangrass. Several of these rotation crops may provide a means for depressing M. arenaria population densities on a short-term basis to enhance yields in a subsequent susceptible vegetable crop.     

Relationship Between Crop Losses and Initial Population Densities of Meloidogyne arenaria in Winter-Grown Oriental Melon in Korea

To determine the economic threshold level, oriental melon (Cucumis melo L. cv. Geumssaragi-euncheon) grafted on Shintozoa (Cucurbita maxima × Cu. moschata) was planted in plots (2 × 3 m) under a plastic film in February with a range of initial population densities (Pi) of Meloidogyne arenaria. The relationships of early, late, and total yield to Pi measured in September and January were adequately described by both linear regression and the Seinhorst damage model. Initial nematode densities in September in excess of 14 second-stage juveniles (J2)/100 cm³ soil caused losses in total yields that exceeded the economic threshold and indicate the need for fosthiazate nematicide treatment at current costs. Differences in yield-loss relationships to Pi between early- and late-season harvests enhance the resolution of the management decision and suggest approaches for optimizing returns. Determination of population levels for advisory purposes can be based on assay samples taken several months before planting, which allows time for implementation of management procedures. We introduce (i) an amendment of the economic threshold definition to reflect efficacy of the nematode management procedure under consideration, and (ii) the concept of profit limit as the nematode population at which net returns from the system will become negative.     

Morphological and Morphometrical Characterization of Meloidogyne incognita from Different Host Plants in Four Districts of Punjab,India

The population of M. incognita, the root knot nematode (RKN) was found infesting five different host plants (okra, banana, sunflower, bottle gourd, and brinjal) out of 24 examined from four districts of Punjab, India (Gurdaspur, Ludhiana, Patiala, and Hoshiarpur). Morphological and morphometrical characterization indicated that in the case of mature female, the characters of body length and width, neck length, ratio ‘a’, anus to tail terminus (ATT), interphasmid distance (IPD), and perineal pattern were recorded as stable characters. These taxonomic characters can be reliable for identification. All characters of second-stage juvenile (J2) such as body length, stylet length, head to median bulb length (H-MB), distance from median bulb to excretory pore (MB-EP), tail length, anal body width (ABW), and ratios C and C’ were highly variable. Analysis of interpopulation morphometric characters of mature female of M. incognita, namely, body length, width, and ratio ‘a’ were moderately variable characters (CV 0.26% to 20%) and stylet length, neck length, length of median bulb (LMB), and width of median bulb (WMB) were highly variable (CV 1.0% to 36.1%). In the perineal pattern, the two characters ATT and IPD were moderately variable (CV 8.8% to 17.6%) and two characters, anus to vulval slit (AVS) and length of vulval slit (LVS), were highly variable (CV 2.1% to 40.5%). In J2, body length, stylet length, H-MB, MB-EP, ABW, tail length, ratios C, and C’ were highly variable characters (CV > 12%).     

Sensitive PCR Detection of Meloidogyne arenaria, M. incognita, and M. javanica Extracted from Soil

We have developed a simple PCR assay protocol for detection of the root-knot nematode (RKN) species Meloidogyne arenaria, M. incognita, and M. javanica extracted from soil. Nematodes are extracted from soil using Baermann funnels and centrifugal flotation. The nematode-containing fraction is then digested with proteinase K, and a PCR assay is carried out with primers specific for this group of RKN and with universal primers spanning the ITS of rRNA genes. The presence of RKN J2 can be detected among large numbers of other plant-parasitic and free-living nematodes. The procedure was tested with several soil types and crops from different locations and was found to be sensitive and accurate. Analysis of unknowns and spiked soil samples indicated that detection sensitivity was the same as or higher than by microscopic examination.     

Persistence and Suppressiveness of Pasteuria penetrans to Meloidogyne arenaria Race

The long-term persistence and suppressiveness of Pasteuria penetrans against Meloidogyne arenaria race 1 were investigated in a formerly root-knot nematode suppressive site following 9 years of continuous cultivation of three treatments and 4 years of continuous peanut. The three treatments were two M. arenaria race 1 nonhost crops, bahiagrass (Paspalum notatum cv. Pensacola var. Tifton 9), rhizomal peanut (Arachis glabrata cv. Florigraze), and weed fallow. Two root-knot nematode susceptible weeds commonly observed in weed fallow plots were hairy indigo (Indigofera hirsuta) and alyce clover (Alysicarpus vaginalis). The percentage of J2 with endospores attached reached the highest level of 87% in 2000 in weed fallow, and 63% and 53% in 2002 in bahiagrass and rhizomal peanut, respectively. The percentage of endospore-filled females extracted from peanut roots grown in weed fallow plots increased from nondetectable in 1999 to 56% in 2002, whereas the percentages in bahiagrass and rhizomal peanut plots were 41% and 16%, respectively. Over 4 years, however, there was no strong evidence that endospores densities reached suppressive levels because peanut roots, pods, and pegs were heavily galled, and yields were suppressed. This might be attributed to the discovery of M. javanica infecting peanut in this field in early autumn 2001. A laboratory test confirmed that although the P. penetrans isolate specific to M. arenaria attached to M. javanica J2, no development occurred. In summary, P. penetrans increased on M. arenaria over a 4-year period, but apparently because of infection of M. javanica on peanut at the field site root-knot disease was not suppressed. This was confirmed by a suppressive soil test that showed a higher level of soil suppressiveness than occurred in the field (P ≤ 0.01).     

Additive Effects of Meloidogyne arenaria and Sclerotinin rolfsii on Peanut

Field observations have suggested that infection of peanut by Meloidogyne arenaria increases the incidence of southern blight caused by Sclerotium rolfsii. Three factorial experiments in microplots were conducted to determine if interactions between M. arenaria and S. rolfsii influenced final nematode population densities, incidence of southern blight, or pod yield. Treatments included four or five initial population densities of M. arenaria and three inoculum rates of S. rolfsii. Final nematode population densities were affected by initial nematode densities in all experiments (P = 0.01) and by S. rolfsii in one of three experiments (P = 0.01). Incidence of southern blight increased with increasing inoculum rates of S. rolfsii in all experiments and by the presence of the nematodes in one experiment (P = 0.01). Pod yield decreased with inoculation with S. rolfsii in all experiments (P = 0.05) and by M. arenaria in two of three experiments (P = 0.05). In no experiment was the interaction among treatments significant with respect to final nematode population densities, incidence of southern blight, or pod yield (P = 0.05). The apparent disease complex between M. arenaria and S. rolfsii on peanut is due to additive effects of the two pathogens.