Studies on the epidemiology and control of cucumber black root rot caused by Phomopsis sclerotioides in Iwate Prefecture

Black root rot of cucumber (Cucumis sativus) caused by Phomopsis sclerotioides is a serious greenhouse disease in many countries. P. sclerotioides is soil-borne and infects cucurbit vegetables. Since 2002, the disease has been observed in outdoor-cultivated cucumber grafted on pumpkin rootstock in Iwate Prefecture, Japan, causing severe economic losses. However, epidemiological information on this pathogen is limited, and an effective control method for outdoor-cultivated cucumber is warranted. The four main objectives of this study were as follows: (1) to describe the occurrence of cucumber black root rot and the extent of damage caused by P. sclerotioides in Iwate Prefecture; (2) to investigate the characteristics of the pathogen and its epidemiology; (3) to develop control methods for cucumber black root rot by using a resistant rootstock and/or chloropicrin fumigants; and (4) to evaluate the control method against cucumber black root rot through the application of soil pH amendments by using a converter slag. The results obtained from this study are briefly described below. 1. The occurrence of cucumber black root rot and the extent of damage caused by P. sclerotioides in Iwate Prefecture Black root rot on outdoor-cultivated cucumber grafted on pumpkin rootstock was found in three out of the 58 municipalities of Iwate Prefecture in 2002. Since then, the incidence of black root rot in cucumber crops has increased, affecting 16 out of the 33 amalgamated municipalities in 2012 that contain 29 ex-municipalities. The disease causes sudden wilting of plants, usually immediately before the harvest period, thus resulting in severe losses in the marketable yield. At first, infected plants show slight wilting during periods of high evapotranspiration during the day, although these show recovery at night. Once wilting is observed, most of the infected plants die within few days. In fully wilted plants, the roots become brown to black coloration and finally rot. In addition, most of the rootlets and root hairs fall off. At the last stage of the disease, pseudomicrosclerotia and pseudostromata form on the radicular surface of the plants. Weather conditions largely influence the development and progression of the disease. Wilt symptoms tend to be more severe during below-average summer temperatures such as that during 2003 compared to extraordinarily hot summers such as that in 2010. It has been suggested that high temperatures generally weaken the pathogen and below-average summer temperatures induce plant stress. The disease is frequently found in continuously cropped cucumber fields, as well as in new fields. The results of this study suggest that the pathogen has further spread across Iwate Prefecture, possibly through the transport of contaminated soil by footwear, plant materials, or machinery. Therefore, it is important to prevent the transfer of infested soil and contaminated equipment to uninfested fields. The results of the survey questionnaire collected from 2007 to 2011 revealed that only approximately 40% of cucumber farmers have engaged in fumigation of the infested fields by using chloropicrin, whereas farmers of approximately 60% of infested fields have continued cucumber cultivation without any disinfestation treatment. Sudden wilting was caused not only by cucumber black root rot but also by other plant diseases and pests. Sudden wilting caused by other plant diseases and pests were attributed to the following factors: (1) superinfection with Cucumber mosaic virus (CMV) and Zucchini yellow mosaic virus (ZYMV); (2) Gummy stem blight; (3) Fusarium wilt; (4) Phytophthora rot; (5) root knot; and (6) Monosporascus root rot. To the author's knowledge, for the first time in Japan, Monosporascus root rot of cucumber has been confirmed. This disease was observed in the summer to autumn cucumber harvest period of 2006 in the open fields of Iwate Prefecture. The causal fungus was identified as Monosporascus cannonballus, based on its morphological features and confirmed using polymerase chain reaction (PCR) with a M. cannonballus-specific primer pair. Furthermore, the unique root symptom included the presence of perithecia in the brown lesions; thus, it is easy to distinguish Monosporascus root rot from Phomopsis black root rot. Monosporascus root rot exclusively occurred in cucumber roots and was not found in those grafted on pumpkin rootstocks. 2. Characteristics and epidemiology of P. sclerotioides Cultural characteristics of P. sclerotioides were as follows: (1) the fungus grew on potato dextrose agar (PDA) at a temperature range of 10-30 degC, but did not thrive above 32.5 degC (optimal range: 20-25 degC); (2) in vitro mycelial growth of the pathogen was optimal at a pH range of 4.0-5.0 and decreased at higher or lower pH levels; and (3) the fungus was killed after incubation at 35 degC for 5-7 days, at 37.5 degC for 4-5 days, and at 40 degC for 2-4 days. Disease development of cucumber black root rot was as follows: (1) temperatures between 20 and 25 degC were conducive to disease development, whereas at 30 degC, it was suppressed; (2) symptoms of wilting were enhanced by drought; (3) the disease could occur by inoculation at an extremely low density, even at 0.01% (w/w) of the naturally infested soil; and (4) in most cases, the pathogen could thrive up to a soil depth of at least 40 cm in naturally infested fields. Long-term survival of pseudostromata and pseudomicrosclerotia on the hypocotyl or radicular residual tissues does not occur in dry soil conditions, whereas humid conditions facilitate its long-term survival in soil. It has been previously shown that pseudostromata and pseudomicrosclerotia may play important roles as survival structures in the soil. 3. Control method by using resistant rootstock and/or chloropicrin fumigants against cucumber black root rot To prevent cucumber black root rot, several Cucurbitaceae plants were examined as resistant rootstocks for grafting cucumber plants, and Cucurbita ficifolia and Benincasa hispida were found to be effective. C. ficifolia is highly compatible to cucumber and is easy to graft because of its thick hypocotyl. On the other hand, some cultivars of B. hispida showed inadequate adaptability for grafting cucumber plants. Thus, C. ficifolia can be used as the resistant rootstock for grafting cucumber. However, C. ficifolia, a bloom type rootstock, cannot be extensively used as the resistant rootstock for cucumber grafting in commercial fields because the Japanese market preference is for bloomless fruits. Soil fumigants such as chloropicrin (298.5 L a.i./ha), dazomet (294 kg a.i./ha), metham sodium (180 L a.i./ha), and fluazinam (15 kg a.i./ha) were evaluated for efficacy in naturally infested fields. On the basis of the significant reduction of sudden wilting, chloropicrin was selected for subsequent experiments and used to develop an effective control scheme. A high level of efficacy against cucumber black root rot was observed in all chloropicrin dosage forms: chloropicrin solution (product name: Chloropicrin), chloropicrin emulsion (Chlopic-flow), chloropicrin tablet (Chloropicrin jouzai), and chloropicrin tape (Chlopic-tape). Partial fumigation (injection into polyethylene-mulched beds) resulted in its adequate control, which was better than that by overall fumigation in the fields. After partial fumigation, it was essential to position the cucumber seedlings in the center of the beds because the fumigation process only disinfected the bed interior. For chloropicrin injection into polyethylene-mulched beds, 90-cm-wide beds were found to be more effective than the commonly used 60-cm-wide beds. Since the 90-cm-wide beds represented wider disinfested area than that by the 60-cm-wide beds, the author concluded that the control efficacy was high, but the treatments did not completely control the occurrence of sudden wilt. Combining the treatments with the use of the resistant rootstock C. ficifolia reliably decreased the incidence of sudden wilt, resulting in a 100% reduction in the three experiments compared to the untreated plots. The efficacy of each control measure varied, and thus, integrated methods are considered effective. The combination of the chloropicrin fumigation, polyethylene-mulched beds, and root restriction treatments was also effective because plant roots could grow in sterilized soil with root restriction treatments. However, the combination resulted in yield losses, because root restriction treatments induced stress in the cucumber plants. Therefore, improvement on the practical use of this combination is necessary. Soil fumigation by using chloropicrin (298.5 L a.i./ha) in deeper soil layers (approximately 30-cm depth) resulted in the adequate control of cucumber black root rot. In general, chloropicrin is directly introduced to the soil (at a 15-cm depth) and then covered with a plastic film to suppress its volatilization. In contrast, the chloropicrin deep-injection method is a very simple task, involving only soil surface compaction with a roller to seal off the air spaces between soil particles. Thus, this technique could also be regarded as labor-effective. However, this approach requires its registration as a new agricultural chemical (application expansion) because it is different from the currently used chloropicrin applications in Japan. 4. Control of cucumber black root rot through soil pH amendments by using a converter slag The results of seedling experiments and greenhouse tests showed that the converter slag (product name: Tenro Sekkai, a soil acidity amelioration material, which is a by-product of steelmaking) treatment of infested soil could prevent cucumber black root rot. Furthermore, this approach could significantly reduce the severity of disease while keeping soil pH levels high enough. However, physiological disorders were observed after this treatment in high-pH level soil (pH above 8.0; Haplic Andosols). The results suggest that the application of the converter slag to infested fields and maintenance of the soil pH at around 7.5 was optimal for the control of cucumber black root rot. The cucumbers grafted on pumpkin rootstock showed remarkable black root rot control after converter slag treatment, although sufficient effect was not seen in non-grafted cucumbers. Commercial field tests using cucumbers grafted on bloomless-type rootstock (Cucurbita moschata) showed that the technique could significantly reduce the severity of disease at a soil pH of 7.5 and a 10-cm amended soil depth (19,600 to 38,000 kg/ha, pH 7.5). No physiological disorders were observed in the cucumbers collected from six field tests that were conducted between 2009 and 2011. For this approach, a soil depth of 10 cm is recommended based on the cost of the treatment and its control-effectiveness. In the next section, the factors responsible for disease suppression, calcium dosage, and soil pH elevation are described. The addition of CaSO4 (soil pH did not change) to infested soil did not generate the same effect on seedling survival as that seen with CaCO3 or the converter slag (containing approximately 41.4% CaO) approaches. The results suggest that the elevation of soil pH, and not calcium supplementation of the soil, was responsible for disease suppression.