The aim of this study was to evaluate the effect of different rotation crops used by nurseries on BFD and CRR pathogens by detecting and quantifying these pathogens in nursery plants and soil.

 

Introduction

Grapevine decline is a disease complex that affects young vines in all grape growing regions of the world and leads to a lower survival rate for young vines. A major contributing factor to grapevine decline is black foot disease (BFD), and crown and root rot (CRR). Studies in South Africa have shown that BFD and CRR pathogens are present in nursery vines as asymptomatic infections. Black foot disease of grapevines is a serious soilborne disease occurring mainly in nurseries and young vineyards, and are more often encountered at disease clinics (Department of Plant Pathology, SU and ARC Infruitec-Nietvoorbij) than CRR.

Black foot disease of grapevines is caused by species of the genera Dactylonectria (previously known as Cylindrocarpon), Ilyonectria (previously Cylindrocarpon), Campylocarpon, Cylindrocladiella and Thelonectria. The BFD species are soil inhabitants and are regarded as saprobes and weak pathogens, and infect roots through wounds and natural openings. In nurseries, the most visible symptoms include plants with reduced vigour, shortened shoots and internodes, sparse foliage and interveinal chlorosis. Upon dissection of the roots and basal ends of rootstocks, black discolouration, gum inclusions of the xylem vessels, and brown vascular streaking can be observed. A reduction in root biomass and root hairs, and sunken necrotic root lesions may also be observed on afflicted plants. In young vines (less than five years old) death can occur rapidly. All BFD pathogens except Campylocarpon species can produce thick-walled chlamydospores which would allow these pathogens to persist in the soil for long periods.

Crown and root rot is caused by species belonging to the genera Phytophthora, Pythium and Phytopythium. Root and crown rot symptoms include black to brown root necrosis, while the above ground plant parts show typical decline symptoms, such as stunted growth, leaf chlorosis, wilting, die-back, poor fruit-set and shoot growth. It has been reported that some Phytophthora spp. may even infect the crowns and cause cankers which may girdle grapevine trunks. This girdling may then lead to plant collapse. The infected vines may be isolated or occur in small groups. CRR pathogens can produce sexual oospores that can persist in the soil.

Nursery soils in South Africa are known as an inoculum source for BFD and CRR pathogens. The treatment of BFD is limited to cultural practices, such as hot water treatment, as there are no fungicides registered against this disease. However, the treatment for CRR involves cultural practices, as well as the use of phosphonate fungicides. The extent to which BFD and CRR pathogens are present in nursery soils is not known. Also the influence of rotation crops on the occurrence of BFD and CRR has not been investigated, and could hold potential in an integrated control strategy.

 

The objectives of the study were:

  • To adapt existing molecular quantification methods to directly quantify BFD and CRR pathogen DNA in grapevine nursery soils over a three-year period, and
  • To isolate these pathogens from grapevines, weeds and rotation crops in these nurseries over the same three-year period.

 

Methodology and results

In 2013, soil and grafted grapevine cuttings were collected from five nurseries. In 2014, rotation crops and weeds were collected from the same nurseries at approximately the same sites within the nursery. In 2015, soil and grapevine samples were collected again from the five nurseries. The soil samples were taken at two depths (0 – 30 cm and 30 – 60 cm), 10 cm away from the grapevines that were sampled (Picture 1). Soil analyses were conducted on the soil sampled.

 

Objective 1: Quantify BFD and CRR pathogen DNA in grapevine nursery soils

Total DNA extractions were done on the soil samples. The pathogen DNA in these total soil DNA samples were then quantified using optimised Quantitative Real-Time Polymerase Chain Reaction (qPCR) protocols for the detection of Phytophthora species, Pythium irregulare, and Dactylonectria and Ilyonectria species. qPCR is a molecular technique used to detect and quantify target DNA in a sample (soil), while the DNA amplification can be viewed in real-time.

Dactylonectria and Ilyonectria species, Py. irregulare and Phytophthora DNA was successfully detected, and quantified from grapevine nursery soil. The Dactylonectria and Ilyonectria DNA was detected in all nursery soil samples, except for one site in one nursery, and ranged from 0.04 pg.μL-1 to 37.14 pg.μL-1. The Py. irregulare was detected in all nurseries (though not for all sites with 22 of the sites testing negative over the three-year period of sampling) with the DNA concentration in the soil that ranged from 0.01 pg.μL-1 to 3.77 pg.μL-1. Phytophthora DNA was detected in all samples except for three sites in one nursery in 2013. The Phytophthora DNA concentrations ranged from 0.01 pg.μL-1 to 29.53 pg.μL-1. Pathogen DNA concentrations increased over the three years for four of the nurseries.

 

PICTURE 1. Soil samples were taken with an auger. Nursery soils were different as can be seen from a dryer soil (a) versus a more waterlogged soil (b).

 

Objective 2: Isolate BFD and CRR pathogens from grapevines, weeds and rotation crops from nurseries

Pathogen (fungal and Oomycete) isolations were done from the grapevine cuttings, weeds and rotation crops. Isolations were made from the roots and vascular tissue of the grapevines onto potato dextrose agar, and Pythium and Phytophthora selective media. The fungal and Oomycete cultures were identified using Polymerase Chain Reaction (PCR), and sequencing of two gene regions (ITS-rDNA and histone H3).

A total of 176 BFD and 108 CRR pathogen isolates were obtained. The predominant BFD pathogens isolated from grapevines in these nurseries were Dactylonectria macrodidyma, Campylocarpon pseudofasciculare, and Ca. fasciculare. Symptoms associated with BFD could be seen on the vines isolated from (Picture 2). Other BFD pathogens, such as D. novozelandica, D. toressensis, D. alcacerensis, D. pauciseptata and I. liriodendri, were isolated as well. The predominant CRR pathogens were Pythium irregulare followed by Phytopythium vexans. The other CRR pathogens isolated included Pythium sylvaticum, Py. ultimum, Py. heterothallicum, Py. rostratum, and Phytophthora niederhauserii.

 

PICTURE 2. Isolations were made from visually healthy vines. A longitudinal cut through the base of the vine clearly show the typical brown discolouration due to infection of black foot pathogens (a). A transverse cut through the rootstock shows brown water soaked discolouration associated with black foot (b).

 

The rotation crops sampled included Canola, white mustard, forage radish, Triticale and lupines. The weeds sampled included Johnson grass, rye grass, winter grass, Cape marigold and corn spurry. No pathogens were isolated from Canola, white mustard and lupines. Of the different rotation crops Triticale had the greatest diversity of pathogens. Four isolates of Py. irregulare, two isolates of Py. ultimum, D. pauciseptata, D. novozelandica, and one isolate of D. macrodidyma were obtained from Triticale roots. One isolate of Py. irregulare was obtained from forage radish. Pathogens were also isolated from the weeds corn spurry, Cape marigold, rye grass, winter grass and Johnson grass. One isolate of D. macrodidyma was obtained from the weed, corn spurry, while one and two isolates of Py. irregulare were obtained from the weeds, winter grass and rye grass, respectively. Two Py. ultimum isolates were also obtained from rye grass and Cape marigold.

 

Discussion

No correlations could be made between DNA concentrations and occurrence of pathogens in vines or rotation crop. However, one nursery had no pathogens present in the vines isolated from, and the concentration of the Dactylonectria and Ilyonectria DNA was significantly less than two other nurseries for all three years. This nursery is situated on the slopes of a hill and makes use of ridging as a method to improve soil drainage. Other factors that could also contribute to this, could be that it does not have a long history of grapevine nursery cultivation and implements a three-year rotation system, whereas two-year rotation systems were used in the other nurseries sampled.

The soil characteristics determined by the laboratory tests were all considered to be within the normal ranges (Dr. F. Ellis, personal communication). Therefore, stress as a result of nutrient deficiencies or salinity were deemed to be negligible. The soil wetness index is an important characteristic which indicate the potential of a soil to become waterlogged. Four of the nurseries are situated in a production area with low lying alluvial soils which is prone to wetness and also had higher soil wetness index. These soil conditions can contribute to the occurrence of BFD or CRR pathogen infections.

The presence of the pathogen DNA in the soil, as well as in grapevine plants, shows that pathogen inoculum persists in the soil during the crop rotation year. The DNA concentrations in the crop rotation year (2014) for Dactylonectria and Ilyonectria species, as well as Phytophthora species, were equal or higher than in the first grapevine year (2013) investigated. This, together with the fact that the DNA concentrations for these pathogens were higher in the second grapevine year investigated (2015), indicate that the pathogens survive successfully in the soil or in the roots and basal ends of specific weeds and rotation crops.

This study has highlighted the possible effects of soil wetness, rotation crops, crop rotation system (two- vs. three-year rotation systems), and cropping history on the incidence of BFD and CRR pathogens in grapevine nurseries in the Western Cape. The complexity of interactions and diversity of factors involved, preclude the drawing of concrete conclusions with regard to crop rotation system to be used to suppress soilborne pathogens. A more extensive study over a longer period of time should be conducted on the suitability of different rotation crops currently used in grapevine nurseries in South Africa and especially the contribution of Brassica crops for biofumigation.

 

Conclusions 

  • This is the first study in South Africa to detect BFD and CRR pathogen DNA from the soil. DNA extraction and qPCR protocols were optimised for soil samples.
  • BFD and CRR pathogen DNA were detected from soil of all five nurseries investigated. From four of the nurseries these pathogens were also isolated from nursery vines collected in year one and year three.
  • Of the rotation crops, only Triticale and forage radish harboured black foot and crown and root rot pathogens, whereas no pathogens were isolated from Canola, white mustard and lupines. The latter rotation crops would be recommended.
  • The weeds corn spurry, Cape marigold, rye grass, winter grass and Johnson grass, harboured pathogens, and may serve as an alternative host in the absence of grapevines (during crop rotation and fallow periods). Weed control would aid in reducing the carry over of pathogens from one vine cropping year to another.
  • Since nursery vines get infected from BFD and CRR pathogens present in nursery soils, the most viable solution is hot water treatment of nursery vines.

 

– For more information, contact Lizel Mostert at lmost@sun.ac.za.

 

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