Distribution and severity of bacterial brown spot on dry beans in South Africa : An update

Bacterial brown spot (BBS) of common bean (Phaseolus vulgaris) is a seed-borne bacterial disease caused by Pseudomonas syringae pv. syringae (Pss). Dry bean is an important field crop in South Africa and all commercially available South African dry bean cultivars are susceptible to BBS. The aim of this study was to determine the current distribution and severity of BBS in the dry bean production areas of South Africa. We surveyed 31 locations in five provinces. Disease severity was evaluated on selected plots on a 0–9 scale in four different farming systems: commercial and subsistence farms and strip and national cultivar trials. Leaves with typical BBS symptoms were harvested and transferred to the laboratory. Bacteria were isolated following standard procedure using King’s B medium. Identification of pure isolates was done using physiological and biochemical techniques. Incidence and severity values were used to calculate a disease index. BBS was observed in 88% of locations. BBS incidence was recorded in 54% to 100% of all the farming systems surveyed during the 2008/2009 and 2010/2011 seasons combined. BBS was more severe in the strip and national cultivar trials than on subsistence and commercial farms during the 2008/2009 season and more severe on the commercial farm and in national cultivar trials than on subsistence farms and strip trials during the 2010/2011 season. Findings of this study signal the importance of developing BBS-resistant dry bean cultivars for South Africa.


Introduction
Bacterial brown spot (BBS), caused by Pseudomonas syringae pv.2][3] The disease is seed-borne and mainly infects foliage and to a lesser extent pods.It is prevalent where dry bean monocropping is practised and spreads through seed transmission, wind and human movement from infected to uninfected areas. 4[7] On common bean leaves, BBS symptoms may first appear as water-soaked spots, which gradually enlarge and dry up, and are often surrounded by a narrow yellow or light green zone.Lesions may coalesce and occasionally abscise, subsequently giving the foliage a tattered appearance. 6Water-soaked spots may also appear on infected pods as circular and initially water-soaked spots, later becoming darker green and sunken brown and necrotic, and causing the infected area to bend as a result of cessation of growth on young pods at the point of infection. 6ccasionally, ring spots of lesions occur around a central lesion. 8Infected seeds initially have water-soaked spots and later become brown and shrivelled when infection is severe. 9Stem lesions appear when the pathogen develops systemically. 8Sources of inoculum include seed and infected volunteer beans 10 , weed hosts and plant debris 11,12 .
2][13] The pathogen has also been reported in Algeria, Asia, Australia, Egypt, Europe, Ethiopia, Kenya, Lesotho, Malawi, Mauritius, Morocco, New Zealand, Tanzania, Tunisia, Uganda and Zimbabwe. 14,15Although Melis 16 reported the incidence of BBS as being 'only occasional' in South Africa, the disease was first described in 1994 17 when 100% incidence on the cultivar Bonus was recorded.Subsequent reports indicated BBS incidences as high as 93% in seed production fields and 100% in commercial fields, although severities were generally low (≤ 2 on a 1-9 scale). 2 The disease is more prevalent during the reproduction stage, gradually lessening as plants reach physiological maturity.BBS epidemics are favoured by warm humid conditions (>95% RH, 28-32 °C) as well as thunderstorms and hail. 6,10These conditions characterise the South African climate, especially in the central and eastern parts of the country, where dry beans are largely planted on commercial scale.BBS management methods include the use of disease-free seed, application of copper-based fungicides, crop rotation, sanitation and avoiding working the field when wet. 6 Copper-based fungicides only serve as a preventative measure and some Pss populations are resistant to copper sprays. 18,19eld losses of up to 55% associated with BBS have previously been reported in South Africa. 17This loss remains a concern, mainly in commercial farming, because the crop has been reported as an important field crop on account of its nutritional composition 20 and it is a staple food in the eastern parts of the country.Commercial annual dry bean production averages 65 000 tonnes on approximately 50 000 hectares. 21Crops are composed mainly of red speckled sugar (65-75%), small white canning (10-20%) and large white kidney beans (5-10%), and to a lesser extent of alubia (1-5%) and carioca beans (3-5%). 22Red speckled sugar beans also dominate the composition of beans grown by subsistence farmers.Although previous surveys were conducted in 2002, 2 recent documentation on the incidence of BBS on dry bean production farms and in research trials of South Africa is not available.The objective of this study was to quantify the distribution and severity of BBS on commercial and subsistence farms, as well as in strip and national cultivar trials of South Africa.These trials are planted annually to determine the performances of cultivars in the main bean producing areas.The information obtained from this survey is required to justify the necessity to embark on a BBS-resistance breeding programme and the deployment of effective means of BBS control in dry bean fields.

Disease surveys in commercial fields
Bacterial brown spot was surveyed in commercial dry bean fields from February to April during the 2008/2009 and 2010/2011 growing seasons to determine incidence and severity.A total of 32 fields at 20 localities (Table 1) was surveyed during flowering, early-and advancedpod stages.The incidence of plants showing typical BBS symptoms was assessed in 10 randomly selected groups of 10 consecutive plants amounting to 100 plants/field.Incidence and severity values were used to calculate a disease index (D I ) using the model: where I is the incidence of diseased plants (%), S is the mean severity of foliar symptoms and M is the maximum severity value (i.e. 9). 23

Disease surveys in strip trials
Three strip trials were surveyed in March during the 2008/2009 and 2010/2011 dry bean growing seasons to determine BBS incidence and severity (Table 2).For each cultivar, 10 randomly selected plants were assessed for incidence and severity and D I was calculated as described above.

Disease surveys in the national cultivar trials
A total of 22 cultivar trials was surveyed for the incidence and severity of BBS at 12 localities during the 2008/2009 and 2010/2011 dry bean growing seasons (Table 3).The plants were evaluated as described above.Samples were collected from only severely infected plants.

, disease index (expressed as % infected leaves); I, incidence of diseased plants (expressed as % infected plants); S, mean severity of foliar symptoms (expressed as % infected leaf area).
Disease severity was evaluated on a 1-9 scale, where 1=0% foliage affected, 3=2% foliage affected, 5=5% foliage affected, 7=10% foliage affected and 9=25% foliage affected. 24solation and identification of bacterial brown spot pathogen A total of 378 diseased leaf samples were collected and used to isolate the BBS pathogen, Pss.Leaves were rinsed under running tap water for approximately 10 min, surface sterilised for 3 min in 3.5% sodium hypochlorite and rinsed twice in sterile water for 1 min at a time.Leaves were macerated in a droplet of sterile water and the macerate was streaked onto King's B agar. 25 Plates were incubated for 48-72 h at 25 °C.
Fluorescent colonies typical of Pseudomonas spp.were selected under UV light and incubated for 48 h on King's B medium for purification.Isolates were tested for oxidase (-) and levan production (+). 26Carbon source utilisation of sucrose, mannitol, sorbitol and inositol was used to distinguish Pss from P. syringae pv.phaseolicola (Psp) isolates. 27solates were subjected to the LOPAT test, 28 the analytical profile index 20E (bioMerieux, Marcy l'Etoile, France) and Biolog GN Microplate (Biolog, Hayward, CA, USA) analyses.Agglutination of Pss-specific antiserum antibody (Express Kit, NEOGEN Europe Ltd., Scotland, UK) confirmed the identity of isolates.

Commercial fields
Results from surveys conducted in commercial fields are given in Table 1.The BBS incidence, severity and disease index on commercial farms ranged from 0% to 15%, 0% to 3%, and 0% to 7.5%, respectively, during the 2008/2009 dry bean growing season (Table 1).During the 2010/2011 dry bean growing season, BBS incidence, severity and index ranged from 4% to 9%, 1% to 3%, and 2.7% to 9%, respectively.The disease was most severe at Warden (3%) and Estcourt (3%) during the 2008/2009 season and at Warden (3%) and Sterkfontein (3%) during the 2010/2011 dry bean growing season.Generally, BBS occurred on 96% of commercial farms during the two seasons.

Strip trials
Results from surveys conducted in strip trials are given in Table 2.During the 2008/2009 dry bean growing season, one strip trial was surveyed, in which BBS incidence was 22%, severity 3% and the disease index was 16.5%.Two strip trials in two localities were surveyed during the 2010/2011 dry bean growing season in which BBS incidence ranged from 0% to 7%, severity from 0% to 2% and the index from 0% to 7%.Of the two seasons surveyed, the disease was most severe at Vryheid (3%) during the 2008/2009 season.BBS was not recorded in the strip trial surveyed in Grootpan.BBS occurred in 67% of strip trials during the two seasons.

Subsistence farms
Results from surveys conducted on subsistence farms are given in Table 4. BBS incidence, severity and index ranged from 0% to 7%, 0% to 2%, and 0% to 7%, respectively, during the 2008/2009 dry bean growing season.BBS incidence, severity and index ranged from 0% to 8%, 0% to 2%, and 0% to 8% during the 2010/2011 dry bean growing season.BBS was observed in up to 46% of subsistence farms.Ohrigstad (2%) and Mgwagwa (2%), respectively, had the highest disease severity during the Maraxwe 600 0 0 0 --- † Sources: altitude 33-37 -, location not surveyed for that season; D I , disease index (expressed as % infected leaves); I, incidence of diseased plants (expressed as % infected plants); S, mean severity of foliar symptoms (expressed as % infected leaf area).Disease severity was evaluated on a 1-9 scale, where 1=0% foliage affected, 3=2% foliage affected, 5=5% foliage affected, 7=10% foliage affected and 9=25% foliage affected.Pss is one of the few bacterial species with the ability to inhabit the host leaf surface without causing disease. 31However, frost injury can occur because Pss has the ability to catalyse ice formation at temperatures above -5 °C. 32The results discussed here do not take into account epiphytic cells that could have been present on the leaf surfaces, even on leaves appearing healthy.
Low disease severities could also be attributed to the fact that many commercial farmers plant disease-free seed.As Pss is seed-borne, it continues to be a threat because a number of producers, mainly subsistence farmers, still plant their own seed, which can impact the spread of the disease in seasons in which conditions are conducive for the spread of disease.
The findings of this study indicate recurring incidences of high proportions in commercial farms, as reported previously. 2 High BBS incidences in strip and national cultivar trials and in subsistence farms and home gardens were also high, which could be attributed to continuous use of contaminated seed.The current BBS incidences and severities signify the importance for the development, through backcross breeding, of BBS-resistant dry bean cultivars if incidences and severities of greater proportions are to be avoided, thereby ensuring sustained dry bean production in South Africa.

Figure 1 :
Figure 1: Dry bean production levels in the six main production provinces of South Africa during the 2008/2009 (left value) and 2010/2011 (right value) growing seasons.

Table 2 :
Occurrence of bacterial brown spot in dry bean strip trials of South Africa

Table 4 :
Occurrence of bacterial brown spot in dry bean subsistence farms of South AfricaWith the exception of the Free State Province, the Limpopo and North West Provinces generally had lower BBS incidences and severities, and are characterised by average annual temperatures (Table5) of at least 18.3 °C.This observation could therefore imply that very high temperatures might contribute to lower BBS incidences and severities.