Fov is divided into races, biotypes and genotypes. Genotypes are distinguished based on genetic variations among isolates. Isolates with morphological or physiological differences without described genetic differences are labeled biotypes (Downie, 2010). The race classification for Fov was originally based on pathogenicity of an isolate to different cotton cultivars or crop species. For example, race 1 caused disease on tobacco but not on soybean, whereas race 2 was capable of infecting both crops (Armstrong and Armstrong, 1958). Fov is genetically diverse and traditionally isolates were classified into eight nominal races, but they are now classified based only on DNA sequence, primarily partial sequence of
In southern Georgia, awareness of FW among farmers, extension agents and consultants has increased greatly in recent years, as have yield losses attributed to this disease (Robert Kemerait, University of Georgia, personal observation). In fields infested with
Soil and plant samples were collected between June and November in 2015 and 2016 from cotton fields showing symptoms of FW. The survey included six counties and 11 fields in 2015 and seven counties and 17 fields in 2016 (Table 1). The Tifton fields 1, 4, and 5 in 2015 are the same as fields 3, 5, and 4 in 2016, respectively. County extension agents provided general information about soil proprieties such as soil texture, previous crop, and cotton varieties planted.
County, cotton variety, soil description, planting date and sampling date for fields in Georgia sampled for
Year | County | Field | Cotton varietya | Soil descriptionb | Planting date | Sampling date |
---|---|---|---|---|---|---|
2015 | ||||||
Ben Hill | 1 | DP 1252 | Leefield loamy sand | May 15 | August 13 | |
Berrien | 1 | DP 1252 | Leefield loamy sand | April 24 | August 28 | |
Cook | 1 | DP 1252 | Stilson loamy sand | May 1 | July 24 | |
Lowndes | 1 | DP 1050 | Tifton loamy sand | May 8 | August 28 | |
Tattnall | 1 | DP 1050 | Loamy sandy | May 20 | Mid-August | |
2 | DP 1050/DP 1137 | Loamy sandy | May 15 | Mid-August | ||
Tift | 1 | DP 1252/PHY 487/DP 1454 | Stilson/Dothan loamy sand | May 4 | June 24 | |
2 | PHY 333 | Ocilla loamy sand | June 2 | August 28 | ||
3 | DP 1252 | Ocilla loamy sand | unknown | July 10 | ||
4 | DP 1252/DP 1555 | Dothan loamy sand | unknown | September 30 | ||
5 | DP 1454 | Dothan loamy sand | May 7 | June 19 | ||
2016 | ||||||
Coffee | 1 | ST 6182 | Stilson loamy sandy | May 15 | August 30 | |
Cook | 1 | DP 1252 | Leefield/Irvington loamy sand | May 5 | August 26 | |
Colquitt | 1 | DP 1538 | Leefield loamy sand | April 20 | August 31 | |
2 | DP 1252 | Dothan loamy sand | 1st week of May | August 31 | ||
3 | DP 1553 | Ocilla loamy fine sand | Mid May | August 31 | ||
4 | unknown | unknown | unknown | December 1 | ||
Tattnall | 1 | DP 1558 | Irvington loamy sand | May 13 | September 22 | |
2 | DP 1553/DP 1558 | Pelham loamy sand | April 27 | September 22 | ||
3 | DP 1553/DP 1558 | Pelham loamy sand | April 29 | September 22 | ||
Tift | 1 | DP 1252 | Ocilla loamy sand | April 24 | August 24 | |
2 | DP 1252 | Dothan/Fuquay/ Tifton loamy sand | April 26 | August 24 | ||
3 | DP 1252 | Stilson/Dothan loamy sand | April 23 | August 24 | ||
4 | ST 5115/ST 6182 | Fuquay/Tifton loamy sand | June 1 | August 26 | ||
5 | DP 1558 | Dothan loamy sand | May 15 | August 26 | ||
Ware | 1 | PHY 444 | Leefield loamy sand | May 18–20 | September 7 | |
2 | DP 1558 | Pelham loamy sand | May 16 | September 7 | ||
Worth | 1 | DP 1558 | Fuquay loamy sand | May 7 | September 21 |
In total, 8 to 15 samples were collected from each field. For each sample, a single plant showing symptoms of FW was arbitrarily selected, carefully uprooted, and the roots were observed for the presence or absence of root-knot nematode galls. The soil associated with the plant’s root system and a piece of the diseased plant’s stem from near the base were sealed in a plastic bag. The samples were stored at 10°C until processing. Nematodes were extracted from 150 cm3 of soil for each sample by the centrifugal flotation method (Jenkins, 1964), and the number of individuals for each genus of plant-parasitic nematode present was recorded.
For 2015 samples, Fov was isolated by disinfesting infected stem pieces in 0.875% NaOCl for 1 min, and then placing them into Komada selective medium (Komada, 1975) for five days. Five stem pieces per plant were cut open longitudinally and placed on a single plate. Individual colonies were then selected and transferred to a new plate containing Komada medium and allowed to grow for seven days. Then, single-spore isolations were made through successive serial dilution of conidial suspensions.
DNA was extracted separately from at least three different single-spore isolates per field. DNA extraction was accomplished by inoculating 125 ml flasks of potato dextrose broth with a mycelial plug (0.7 cm diameter) from the single-spore isolates and allowing them to grow for four days on a rotational shaker at 100 rpm. All the mycelium was then dried overnight on a sterile plate prior to using the modified DNA extraction method of Abd-Elsalam et al. (2003). Mycelium was macerated in liquid nitrogen and then put into 1.5 ml Eppendorf tubes prior to adding extraction buffer (200 mM Tris pH = 8.5, 250 mM NaCl, 25 mM EDTA, and 0.5% SDS). The sample was mixed on a vortex mixer and 4 µl of RNAase was added prior to incubation in a water bath for 65°C for 10 min. Then, 130 µl of 3 M sodium acetate (pH = 5.2) was added and the tubes were held at −20°C for 10 min. After centrifuging the sample for 15 min at 4,000 rpm, a 400 µl aliquot of the supernatant was collected and transferred to a 1.5 ml Epperndorf tube. The sample was then mixed in a 1:1 ratio with chloroform for 3 min and then centrifuged for 10 min at 8,000 rpm. The supernatant was removed and 650 µl of isopropanol was added to the tube to precipitate the DNA. Isopropanol was removed after centrifuging tubes for 10 min at 4,000 rpm. Finally, 300 µl of 70% ethanol was added into the tubes and centrifuged for 1 min at 4,000 rpm. Ethanol was removed from the tube and DNA allowed to dry overnight. Deionized water (100 µl) was added to rehydrate the DNA. The translation elongation factor (EF–1α) and IGS regions were PCR-amplified and sequenced for the Fov isolates as described by Kim et al. (2005) and Cianchetta et al. (2015). PCR reactions consisted of 12.5 μl of 2X Mean Green Master Mix (Syzygy Biotech; Grand Rapids, MI), 1 μl of each 10 μM primer and 2 μl of genomic DNA in a 25 μl reaction. Amplification reactions were performed in a thermocycler (PTC–100; MJ Research, Watertown, MA) under conditions as described by Cianchetta et al. (2015). PCR products were purified with a GeneJET PCR Purification Kit (Thermo Fisher Scientific, Inc., Waltham, MA) following the manufacturer’s instructions. A 24 μl sample of 40–68 ng/µl of PCR product and 12 μl of each 3 μM primer were sent to Quintara Biosciences (2600 Hilltop Dr, Building B, R332, Richmond, CA) for sequencing. Sequences were manually checked and aligned with known Fov sequences from GenBank database and private database to identify race or genotype.
For 2016 samples, Fov was isolated by washing cotton stems in soapy water, then surface sanitizing for 1 min in 95% ethanol, 2 min in sodium hypochlorite solution and 1 min in sterile deionized water. The outer layer of bark was removed, and small slivers of stem tissue were plated on acidified quarter-strength potato dextrose agar. Petri plates were incubated at 23°C with a 12-hr photoperiod for six to seven days. Single spores of cultures morphologically resembling
Genomic DNA was extracted from four single-spore isolates per field. Isolates were grown on potato dextrose agar overlain with sterile cellophane for six to seven days, after which time mycelia were harvested and lyophilized. Approximately 50 milligrams of lyophilized mycelia were placed in 2-ml microcentrifuge tubes with sterile glass beads, and macerated into a fine powder in a Geno/Grinder® (SPEX SamplePrep, Metuchen, NJ). DNA was extracted using a DNeasy Plant Mini kit (QIAGEN, Valencia, CA) following manufacturer protocol with the following modification: samples were eluted in 25 μl AE buffer (as opposed to 50 μl as stated in protocol) to increase the final concentration of DNA. PCR amplification, sequencing and genotyping were conducted as previously described.
Fov race 1 was found in all sampled counties in 2015 and 2016. In 2015, no previously unreported races or genotypes were found in Georgia. In addition to race 1, genotype LA108 was found in Lowndes County and LA110 was found in Tift County. In 2016, two new genotypes, LA127/140 and MDS–12, were identified in the fields sampled. Fov races 2 and 8 were found in Lowndes County; race 8 and genotypes LA127/140, LA110, and LA108 were found in Tift County; genotypes LA108 and LA110 were found in Cook County; genotypes LA108, LA110 and MDS–12 were found in Colquitt County; race 2 and genotype LA110 were found in Coffee County; genotype LA108 was found in Ware County; and genotype LA110 was found in Worth and Tattnall Counties (Table 2 and Figure 1).
Fov races and plant-parasitic nematodes found in South Georgia fields in 2015 and 2016.
Fov | Nematode speciesa | |||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
County | Race/genotype description |
|
|
|
|
|
|
|
||||||||
2015 | 2016 | 2015 | 2016 | 2015 | 2016 | 2015 | 2016 | 2015 | 2016 | 2015 | 2016 | 2015 | 2016 | 2015 | 2016 | |
Ben Hill | 1 | ns2 | Y3 | ns | Y | ns | Y | ns | Y | ns | N | ns | N | ns | N | ns |
Berrien | 1 | ns | Y | ns | Y | ns | Y | ns | Y | ns | Y | ns | N | ns | N | ns |
Coffee | ns | 1, 2, LA110 | ns | Y | ns | N | ns | Y | ns | Y | ns | N | ns | N | ns | N |
Cook | 1, LA108, LA110 | 1, LA110 | N | N | Y | Y | N | Y | Y | Y | N | Y | N | N | N | N |
Colquitt | ns | 1, LA110, MDS–12, LA108 | ns | Y | ns | Y | ns | Y | ns | Y | ns | Y | ns | N | ns | Y |
Lowndes | 1, 2, 8, LA108, LA110 | ns | N | ns | Y | ns | Y | ns | Y | ns | Y | ns | N | ns | N | ns |
Tattnall | 1 | 1, LA110 | N | N | Y | Y | Y | Y | N | Y | Y | Y | Y | Y | N | N |
Tift | 1,8, LA110, LA108 | 1,8, LA108, LA110, LA127/140 | Y | Y | Y | Y | Y | Y | Y | Y | Y | N | N | N | N | N |
Ware | ns | 1, 8 | ns | Y | ns | N | ns | Y | ns | Y | ns | N | ns | N | ns | N |
Worth | ns | 1, LA110 | ns | Y | ns | Y | ns | Y | ns | Y | ns | N | ns | N | ns | N |
For the fields included in our survey in 2015, the predominant cotton cultivar planted was DP 1252 B2RF, which is susceptible to
The number of samples for each county in our survey (both years combined) is listed in Table 3. Only samples with one or more of the three species of nematodes reported (
Total number of samples infected with Fov collected from Georgia counties and the respective occurrence of
Samples with concomitant occurrence of nematodes | ||||||
---|---|---|---|---|---|---|
County | Total samples |
|
|
|
All three | |
Ben Hill | 10 |
|
1 | 0 | ||
|
0 | 6 | ||||
|
0 | 1 | 0 | |||
Berrien | 10 |
|
0 | 0 | ||
|
0 | 6 | ||||
|
0 | 1 | 4 | |||
Coffee | 8 |
|
4 | 0 | ||
|
0 | 0 | ||||
|
4 | 0 | 0 | |||
Cook | 23 |
|
0 | 0 | ||
|
0 | 3 | ||||
|
0 | 11 | 5 | |||
Colquitt | 40 |
|
9 | 0 | ||
|
0 | 1 | ||||
|
16 | 8 | 2 | |||
Lowndes | 10 |
|
0 | 0 | ||
|
0 | 6 | ||||
|
0 | 4 | 0 | |||
Tattnall | 50 |
|
0 | 0 | ||
|
0 | 32 | ||||
|
0 | 6 | 0 | |||
Tift | 108 |
|
5 | 11 | ||
|
6 | 28 | ||||
|
4 | 9 | 8 | |||
Ware | 20 |
|
0 | 0 | ||
|
0 | 13 | ||||
|
0 | 7 | 0 | |||
Worth | 10 |
|
0 | 0 | ||
|
1 | 3 | ||||
|
0 | 4 | 2 |
For comparison to the results in our survey, samples from cotton fields in Georgia submitted to the Extension Nematology Laboratory at the University of Georgia from 2013 to 2016 were examined. During those four years,
Race 1 was the predominant race of Fov in South Georgia in previous surveys (Holmes et al., 2009; Cianchetta et al., 2015). Additionally, races 2 and 8 and genotypes LA108 and LA110 also have been identified in Georgia (Holmes et al., 2009; Cianchetta et al., 2015). However, our survey is the first to report the presence of genotypes MDS–12 and LA127/140 in Georgia cotton fields. MDS–12 has been previously found in Alabama, which was the first report of this genotype in the USA (Bennett et al., 2013). Importantly, our survey did not find Fov race 4 or either of the two Australian biotypes, which cause severe FW without
Although the underlying mechanism of how
Another possible explanation for the recent increase in the incidence of FW in Georgia could be a genetic change in Fov allowing it to become more virulent. Fov race 4 is highly virulent on many cotton genotypes (Doan and Davis, 2014), and phylogenic trees have shown that LA110 and LA108 are genetically extremely similar to race 4, which may suggest that they share the high virulence of race 4 (Holmes et al., 2009). However, race 4 is reported to be a root-rot pathotype that caused disease following root-dip assays but not following stem injection and is therefore not considered a vascular-competent pathogen, whereas LA110 and LA108 are reported to be vascular-competent pathotypes that interact with nematode infection to increase disease severity in the field (Bell et al., 2017). The genetic differences between race 4 and genotypes LA108 and LA110 could be associated with differences in virulence or the need to interact with nematodes to cause severe disease under field conditions.
The interaction of Fov with nematodes other than