Identification and Virulence of Five Isolates of Root-Knot Nematode Meloidogyne floridensis on Vegetables.

Meloidogyne floridensis is of particular concern because it reproduces on tomato, pepper, corn, and tobacco cultivars that have resistance to the common tropical root-knot nematode (RKN) species (i.e., Meloidogyne incognita, M. arenaria, and M. javanica). During a survey of 436 randomly selected vegetable fields in Georgia in 2018, 6 M. floridensis-infested fields were found and cultured from single egg-mass isolates on a susceptible tomato (cultivar Rutgers), and speciated using molecular analyses. Five isolates of M. floridensis were identified from collard, cowpea, cucumber, watermelon, and tomato fields by DNA sequence-based identification targeting mitochondrial genes (cytochrome c oxidase subunit II, transfer RNAHis, large subunit ribosomal RNA, and NADH dehydrogenase subunit 5). Two greenhouse trials determined the host preference and reproduction level for each M. floridensis isolate. Evaluations were conducted on Rutgers tomato, a resistant tomato (cultivar Skyway, carrying the Mi-1.2 gene), and vegetable crops associated with the origin of M. floridensis populations. This study confirmed that most associated vegetables, except collards, were good hosts to M. floridensis, having a reproduction factor >1. All isolates were able to reproduce aggressively on the resistant tomato. We found variations among M. floridensis isolates in pathogenicity and reproduction levels on the vegetable crops tested which should be considered when using or developing host resistance.

Gene expression profiling reveals transcription factor networks and subgenome bias during Brassica napus seed development.

We profiled the global gene expression landscape across the reproductive lifecycle of Brassica napus. Comparative analysis of this nascent amphidiploid revealed the contribution of each subgenome to plant reproduction. Whole-genome transcription factor networks identified BZIP11 as a transcriptional regulator of early B. napus seed development. Knockdown of BZIP11 using RNA interference resulted in a similar reduction in gene activity of predicted gene targets, and a reproductive-lethal phenotype. Global mRNA profiling revealed lower accumulation of Cn subgenome transcripts relative to the An subgenome. Subgenome-specific transcription factor networks identified distinct transcription factor families enriched in each of the An and Cn subgenomes early in seed development. Analysis of laser-microdissected seed subregions further reveal subgenome expression dynamics in the embryo, endosperm and seed coat of early stage seeds. Transcription factors predicted to be regulators encoded by the An subgenome are expressed primarily in the seed coat, whereas regulators encoded by the Cn subgenome were expressed primarily in the embryo. Data suggest subgenome bias are characteristic features of the B. napus seed throughout development, and that such bias might not be universal across the embryo, endosperm and seed coat of the developing seed. Transcriptional networks spanning both the An and Cn genomes of the whole B. napus seed can identify valuable targets for seed development research and that -omics level approaches to studying gene regulation in B. napus can benefit from both broad and high-resolution analyses.

First Report of the Root-Knot Nematode, Meloidogyne floridensis, on Tomato in Georgia, USA.

Meloidogyne floridensis, also known as the peach root-knot nematode (RKN), is a new emerging species found to break crop host-resistance to M. incognita (Stanley et al. 2009). It was first described from Florida (Handoo et al. 2004) parasitizing M. incognita-resistant rootstock cultivars of peach (Prunus persica), and tomato (Solanum lycopersicum) (Church 2005). The nematode has recently been reported in California's almond orchards (Westphal et al. 2019) and peach rootstock (cv. Guardian) in South Carolina (Reighard et al. 2019). In a 2018 survey of vegetable fields sampled randomly in South Georgia, RKN was found with a high density (5,264 second-stage juveniles (J2)/100 cm3 of soil) from a tomato field in Ware County, GA. The soil sample consist of 30 soil cores sampled at 20-cm depth across the field in a zig-zag motion. To perform Koch's postulate, 2,000 eggs from a single egg-mass culture were inoculated into deepots filled with mixture of sand and sterilized field soil (1:1 v/v) and grown with tomato cv. Rutgers for 60 days in the greenhouse. A reproduction factor of 21.1 ± 6.1 was obtained confirming the nematode parasitism on tomato (Fig. 1S). For molecular identification, DNA was extracted by smashing three individual females isolated from the galled roots in 50 µl sterile distilled water, followed by a freeze-thaw (95°C, 1 min). Results of PCR analyzes by species-specific primers (Fjav/Rjav, Finc/Rinc and Far/Rar) did not detect the nematode species (Zijlstra et al. 2000). PCR products were obtained and sequenced from two primer sets consisting of the forward NAD5F2 (5'-TATTTTTTGTTTGAGATATATTAG-3') and the reverse NAD5R1 (5'-CGTGAATCTTGATTTTCCATTTTT-3') for amplification of a fragment of the NADH dehydrogenase subunit 5 (NADH5) gene (Janssen et al. 2016), and the forward TRANH (5'-TGAATTTTTTATTGTGATTAA-3') and the reverse MRH106 (5'-AATTTCTAAAGACTTTTCTTAGT-3') for amplification covering a portion of the cytochrome c oxidase subunit II (COII) and large subunit 16SrDNA (16S) gene (Stanton et al. 1997). DNA sequence of NADH5 gene fragment (accession no. MT795954) was 100% identical (532/532 bp) with a M. floridensis isolate from California and South Carolina (accession no. MH729181 and MN072363), while fragment of the COII and 16S genes (accession no. MT787563) was 99.76% identical (421/422 bp) with an isolate from Florida (accession no. DQ228697). The nematode females were also used for morphometric and perennial pattern analysis. Several micrographs with the inverted microscope (ZEISS Axio Vert.A1, Germany) and camera (ZEISS Axiocam 305 color, Germany) were taken from ten J2s for mean, standard deviation and range of body length: 362.7 ± 11.2 (340.4-379) µm, maximum body width: 15 ± 1.3 (12.4-16.4) µm, stylet length: 12.3 ± 1.3 (9.5-14) µm, hyaline tail terminus: 8.9 ± 1.1 (7.5-10.9) µm and tail length: 35.7 ± 4.4 (28.5-39.5) µm. Morphological measurements and configuration of perineal patterns (Fig. 2S) were comparable to previous reports of M. floridensis isolates from Florida (Handoo et al. 2004; Stanley et al. 2009). To the best of our knowledge, this is the first report of M. floridensis in Georgia as the fourth state in the USA after South Carolina, California and Florida. This nematode has been reported to parasitize several vegetable crops, including cucumber, eggplant, tomato, snap bean and squash. Furthermore, RKN resistant cultivars of tomato (harboring Mi-1 gene), pepper (harboring N gene), corn cv. Mp-710 and tobacco cv. NC 95 have been found susceptible to M. floridensis (Stanley et al. 2009), making it a serious threat.

First Report of the Yellow Nutsedge Cyst Nematode, Heterodera cyperi, in Georgia, U.S.A.

Soil samples collected during a survey for plant-parasitic nematodes in Tift County GA in summer 2017 were submitted for routine diagnosis of nematodes to the Extension Nematology Lab at the Department of Plant Pathology, University of Georgia, Athens, Georgia. Cyst nematodes recovered by centrifugal flotation technique were discovered in the samples from two research sites in a field with a history of tobacco and vegetable production. Cyst nematodes from tobacco (10 cysts/100 cm 3 of soil) and vegetable (2 cysts/100 cm 3 of soil) sites had similar morphological features. Morphology and morphometric measurements of the cysts and J2 ( Fig. 1A-C ) were in agreement with those of Heterodera cyperi ( Golden et al., 1962 ; Romero and López-Llorca, 1996 ). Measurements of J2 ( n = 12) included the length (range = 443-494 µm, mean = 467.4 µm) and width (18.3-24.4 µm, 20.6 µm) of body, stylet (19.1-20.8 µm, 20.3 µm), tail (61.6.0-66.4 µm, 64.2 µm), body width at anus (11.9-14.1 µm, 12.8 µm), and hyaline tail terminus (22.7-29.2 µm, 26.3 µm). The lateral field of J2 had three lines. Cysts ( n = 10; Fig. 1C ) were lemon-shaped, light to dark brown in color with protruding neck and vulval cone. The cysts had ambifenestrated vulval cone and no bullae was present. Morphometrics included body length excluding neck (370.5-714.4 µm, 555.7 µm); body width (165.6-411.1 µm, 310.9 µm); neck length (36.5-66.3 µm, 49.8 µm); fenestra length (26.3-42.5 µm, 35.8 µm), and fenestra width (19.1-31.5 µm, 23.8 µm). DNA was extracted from single cysts ( n = 3) and internal transcribed spacer (ITS) of rRNA and partial cytochrome oxidase I ( COI ) genes were amplified with primers TW81/AB28 and Het-coxiF/Het-coxiR, respectively ( Subbotin et al., 2001 ; Subbotin, 2015 ) and sequenced. The resulting sequences were deposited into the GenBank database (Accession no. MG825344 and MG857126) and also subjected to BLAST searches in the database. ITS sequence of H. cyperi showed 100% similarity (100% coverage) with that of a H. cyperi population from Spain (AF274388). COI sequence of H. cyperi showed 89% similarity (98% coverage) with that of H. guangdongensis (MF425735), and 88% similarity (83% coverage) with that of H. elachista (KC618473). The pathogenicity of H. cyperi was examined under greenhouse conditions using tobacco cv. K340, tomato cv. Tribute, cucumber cv. Thunder, and yellow nutsedge ( Cyperus esculentus L.). 3-wk-old seedlings of the test plants were transferred into Deepot D25L cell containers (5-cm-diam. × 25.4-cm deep) filled with sterilized sand: sand: soil mixture (1:2) and then inoculated with 1,000 eggs and J2 of H. cyperi . The plants were grown for 90 d in a greenhouse before examination of roots and extraction of cysts from the soil. Results showed that the nematode failed to reproduce on tobacco, tomato, and cucumber whereas white females and mature cysts of H. cyperi were observed on yellow nutsedge roots ( Fig. 1E ). The results confirmed that yellow nutsedeg was a host for the nematode, and tobacco, tomato, or cucumber were non-hosts. In the United States, H. cyperi was reported from Florida, North Carolina, and Arkansas ( Subbotin et al., 2010 ) infecting Cyperus spp. Yellow nutsedge is considered a serious weed problem in many cropping systems including peanut, cotton, tobacco, and vegetable crops in the Southern United States. To our knowledge, this is the first report of H. cyperi infecting yellow nutsedge in Georgia. Figure 1Photomicrographs of Heterodera cyperi from yellow nutsedge in Georgia. Whole body (A), the anterior region (B), and the posterior region (C) of J2. Cysts (D) recovered from the soil and the vulval cone of cyst with the ambifenestrate fenestra (E). A mature cyst (F) on the surface of yellow nutsedge root infected with the nematode.

An Improved Technique for Sorting Developmental Stages and Assessing Egg Viability of Globodera pallida using High-Throughput Complex Object Parametric Analyzer and Sorter.

The Complex Object Parametric Analyzer and Sorter (COPAS) is a large particle flow cytometer designed for analyzing, sorting, and dispensing objects of varying sizes. We explored the potential of using this instrument to analyze and sort various developmental stages and egg viability of Globodera pallida. Cysts were successfully examined and sorted from debris by optimizing side-scatter and red-fluorescence parameters on the COPAS. We were able to separate eggs and second-stage juveniles from samples of mixed population using extinction and time of flight. Separation of live and dead eggs was examined following staining eggs with SYTOX Green and application of time of flight and green peak height. Data were compared with a commonly used viability assay by which eggs were stained with Meldola's Blue and examined by a microscope. COPAS proved to be effective in assessing viability by detecting two separate gates: live eggs having green fluorescence peaks <190 and dead eggs with the peaks >190. The application of COPAS in combination with SYTOX Green detected a greater number of live eggs than the Meldola's assay, suggesting that SYTOX Green provided an overestimate of live eggs. COPAS noticeably increased the accuracy and reduced the time required for screening and analyzing nematode populations.