Effect of inoculum density and quantitative PCR-based detection of Rhizoctonia solani AG-2-1 and Fusarium avenaceum on canola

Canola seedling blight, caused by Rhizoctonia solani, and Fusarium spp., can result in large yield losses to canola (Brassica napus) at high inoculum pressure. The effect of inoculum density was studied by mixing different amounts of R. solani AG-2-1 and Fusarium avenaceum into a sterilized natural soil and soil-less mix (2:1, v:v) separately, and recording seedling emergence, damping-off and seedling height within ten days after seeding; root rot severity at 12 days after seeding and seed yield at harvest on canola cultivars ‘45H29’ and ‘73-77RR’. Root rot severity increased and emergence, plant height and seed yield decreased with increased inoculum density of both R. solani and F. avenaceum. For quantification of R. solani AG-2-1, a primer and TaqMan probe set (Rs21F/Rs21R/Rs21P) was designed based on the nuclear ribosomal internal transcribed spacer (ITS) region of R. solani AG-2-1. From a conventional PCR amplification, an 88-bp product was amplified from all isolates classified as AG-2-1 with the primers Rs21F and Rs21R. No product was amplified with DNA from isolates belonging to other anastomosis groups of R. solani, other pathogens or the host plant. By using quantitative PCR, DNA amounts as low as 100 fg of R. solani AG-2-1 were detected. The quantity of DNA from soil samples with different inoculum densities estimated using qPCR was highly correlated to the number of colony-forming units (cfu) obtained from the same soil samples for both R. solani AG-2-1 and F. avenaceum.     

Effects of moldboard plowing,chisel plowing and rotation crops on the Rhizoctonia disease of white potato

Two tillage practices, chisel plowing (30 cm) and deep moldboard plowing (22 cm), and five rotation crops (oats, lupine, buckwheat, broccoli and peas) were studied for their effects on the soil population ofRhizoctonia solani AG-3 and on Rhizoctonia disease on potato. All rotation crops were harvested except buckwheat, which was treated as a green manure crop. Chisel plowing significantly reduced (p = 0.05) the incidence and severity of stem lesions on potato caused byR. solani AG-3. In 1990, oats after moldboard plowing significantly increased disease when compared to other crops and broccoli after chisel plowing decreased disease severity. Soil populations ofR. solani AG-3 were significantly lower with chisel plowing. No interactions between tillage and rotation crops were observed. Rhizoctonia solani Kühn is a soil inhabiting plant pathogen found worldwide that affects many plant species including white potato (Solanum tuberosum L.).R. solani attacks potato at one or more stages in development resulting in distinct disease symptoms (25) often termed the Rhizoctonia disease complex of potato. In Maine, only strains AG-3 and AG-5 ofR. solani (4, 5) have been identified as attacking potato and causing four distinct types of symptoms: 1) black scurf (sclerotia) on tubers, 2) stem cankers, 3) aerial tubers and top rosetting, and 4) killing of sprouts. Crop rotation has been reported to reduce the incidence and severity ofR. solani on potato, but no single rotation method controls completely or to a high degree of reliability (11, 23, 24, 26). Deep moldboard plowing has been shown to reduce diseases caused byR. solani andSclerotium rolfsii Sacc. in crops other than potatoes (2, 7, 15, 19, 20). However, Gudmestadet al. (6) reported that deep moldboard plowing increased the severity ofR. solani on stems and stolons of potatoes. The reduction of diseases caused byR. solani andS. rolfsii by moldboard plowing is attributed to low inoculum densities in the upper soil layer by the burial of sclerotia to depths where germination and infection were prevented (14, 15, 19, 20). However, disking to a depth of 5–7 cm did not affect disease as the inoculum remained in the root zone (14, 15). Gurkin (7) states that the rationale for deep moldboard plowing is to promote decay of organic matter, remove organic matter from the infection court and to bury the sclerotia below the infection court. Cultural control techniques are largely preventive and are designed to reduce the quantity or the activity of inoculum by means of crop rotation, tillage practices, green manure crops, etc. (22). This study was conducted to determine individual effects and possible interactions of deep moldboard plowing versus chisel plowing in various rotation crops on the presence ofR. solani AG-3 in the soil and on the incidence of Rhizoctonia disease complex of potato.     

Establishment of Agrobacterium tumefaciens-Mediated Transformation System for Rice Sheath Blight Pathogen Rhizoctonia solani AG-1IA

To construct the T-DNA insertional mutagenesis transformation system for rice sheath blight pathogen Rhizoctonia solani AG-1 IA,the virulent isolate GD118 of this pathogen was selected as an initial isolate for transformation.The conditions for transformation of isolate GD118 were optimized in five aspects,i.e.pre-induction time,co-culture time,acetosyringone(AS) concentration at the co-culture phase,co-culture temperature and pH value of induction solid medium(ISM) at the co-culture phase.Finally,a system ...     

Evaluation ofLaetisaria arvalis as a biological control agent ofRhizoctonia solani on white potato

Laetisaria arvalis, a soil-inhabiting basidiomycete, reducedRhizoctonia solani infection of white potato. A greenhouse study that monitored population levels of both fungi showed that soil infested withL. arvalis suppressed the growth ofR. solani after eight wks. Populations ofL. arvalis andR. solani increased after 11 wks. when both fungi were infested simultaneously and whenL. arvalis was introduced to soil previously infested withR. solani. Five years of field studies showed that variousL. arvalis seed treatments and soil amendments reducedR. solani infection of stems and stolons and sclerotial formation on tubers equal to the standard chemical treatment of pentachloronitrobenzene (PCNB) + thiabendazole. No relationship was shown between populations ofL. arvalis andR. solani in field plot soils, but native or wild strains ofL. arvalis were shown to be present at detectable levels in field soils.     

Anastomosis groups ofRhizoctonia solani on potato in central México and potential for biological and chemical control

Anastomosis group (AG) identity ofRhizoctonia solani isolates collected from potato plants growing near Leon, Guanajuato, in central Mexico was determined. In samples from 15 fields, we found AG-3 and AG-4 with a frequency of 73.5% and 26.5% respectively. AG-4 was found only during the flowering stage of plant growth, whereas AG-3 was present on plants at every stage of development. The efficacy againstR. solani, in vitro andin situ, of certain biocontrol agents (Bacillus subtilis, Gliocladium virens) and fungicides (Pencycuron, Tolcoflos-methyl, Fluazinam, Azoxystrobin) was assessed. CertainBacillus strains collected from the field inhibitedin vitro growth of isolates of certain AGs. Only Pencycuron and Tolcoflos-methyl inhibited AG-3 100%in vitro, whereas in the field, Azoxystrobin and Pencycuron showed the highest efficacy against development of sclerotia on tubers.     

Anastomosis Group and Pathogenicity of <Emphasis Type="Italic">Rhizoctonia solani </Emphasis>Associated with Stem Canker and Black Scurf of Potato in Heilongjiang Province of China

From 2012 to 2015, a total of 226 isolates of Rhizoctonia solani were collected from the stem cankers on potato stems and sclerotia on tubers from different potato cultivation areas of Heilongjiang Province, China. These isolates were assigned to the anastomosis group (AG) by performing conventional PCR assays using previously published primers for ITS-rDNA regions, as well as by observing hyphal interactions where appropriate. Most of the isolates were assigned to AG-3PT (58.85 %), and several were assigned to AG-5 (21.68 %), AG-2-1 (7.08 %) and AG-4 (12.39 %). Pathogenicity tests showed that the AG-3 and AG-5 isolates had the highest virulence, and the disease indices were 1.96a and 2.47a for stem and 1.48a and 1.6a for root (P < 0.05) after analyzed by LSD multiple comparisons, respectively. Both two isolates consistently caused large brown lesions with sunken on the potato stems and roots in in vitro and greenhouse experiments. This is the first detailed report on the AG composition, variability and pathogenicity of R. solani isolates associated with stem cankers and black scurf found on potatoes cultivated in Heilongjiang Province.     

Effect of Brassicaceae seed meals with different glucosinolate profiles on Rhizoctonia root rot in wheat

The soil-borne pathogen Rhizoctonia solani AG 8 causes major yield losses in wheat (Triticum aestivum. L) production worldwide. Plant tissues of Brassicaceae species contain glucosinolates that are hydrolyzed in the presence of the enzyme myrosinase into products with pesticidal properties. Growth chamber studies were conducted to determine the effect of the Brassicaceae seed meals (SMs) from Brassica juncea, Brassica napus and Sinapis alba on the suppression of the R. solani AG 8 infection of winter wheat. Pasteurized sandy soils were amended with intact and denatured SMs of rape seed and mustard at a rate of 0.5% by soil weight. Regardless of the glucosinolate type and content, all intact and denatured Brassicaceae significantly reduced the infection of winter wheat seedlings by R. solani AG 8 compared to the un-amended control. However, soils amended with S. alba SMs had the lowest severity of Rhizoctonia root rot relative to other amended soils. Phytotoxicity arising from the use of Brassicaceae SMs was observed particularly in soils amended with high glucosinolate-containing SMs. These studies demonstrate that Brassicaceae SMs can be used to manage disease caused by R. solani AG-8. However, future studies will need to focus on strategies for diminishing the crop growth-reducing effects associated with Brassicaceae SM amendment to fully maximize these fungicidal benefits.     

Evaluation of thiabendazole and pentachloronitrobenzene for control of the Rhizoctonia disease complex on white potato (Solanum tuberosum L.)

Thiabendazole (TBZ) and pentachloronitrobenzene (PCNB) alone and in combination were laboratory, greenhouse, and field tested during a five-year period (1977-1982) for their effectiveness in reducing incidence and severity of damage caused byRhizoctonia solani to white potato (Solarium tuberosum). Growth ofR. solani on amended potato dextrose agar was prevented at 7 ppm TBZ and 80,000 ppm PCNB. Tuber-borne sclerotial germination and hyphal growth were prevented following seed treatment with 6,000 ppm TBZ, and severely inhibited by combination treatments of 12,500 or 50,000 ppm PCNB and 1,500 ppm TBZ. Seed treatments which provided the most effective field control ofR. solani were combinations of PCNB (50,000, 25,000 ppm) and TBZ (1,500 ppm), followed by TBZ alone (6,000 ppm) and PCNB alone at rates of 50,000 ppm or above.     

Penthiopyrad applied in close proximity to Rhizoctonia solani provided effective disease control in sugar beet

Rhizoctonia solani Kühn is an important pathogen of sugar beet (Beta vulgaris L.) that can cause damping-off and crown and root rot. Commercial cultivars which are highly resistant to the pathogen are not as high yielding as susceptible cultivars under low or absent disease pressure. These resistant cultivars often do not have resistance to other common pathogens such as Aphanomyces cochlioides, Cercospora beticola, and Fusarium oxysporum. Fungicides, such as azoxystrobin which belongs to the quinone outside inhibitors (QoI) class, are necessary for controlling Rhizoctonia solani, but there are concerns about the buildup of fungicide-resistant strains in the targeted pathogen population. There is a need to find effective fungicides from different chemical groups so they can be rotated with the current widely-used azoxystrobin to manage R. solani. The objective of this greenhouse study was to evaluate the efficacy of penthiopyrad, a succinate dehydrogenase inhibitor (SDHI), in managing R. solani on sugar beet using three different application methodologies. Penthiopyrad effectively controlled R. solani on sugar beet when applied at 210, 280, 420, or 550 g a.i./ha in-furrow at planting and as a soil drench at the 4-leaf stage. However, foliar application of penthiopyrad failed to provide disease control. These trials indicated that penthiopyrad needs to be in close proximity or direct contact with R. solani in the soil to provide effective control. Penthiopyrad has the potential to be used as an effective alternate partner with azoxystrobin for controlling R. solani and to help in mitigating the development of fungicide resistant isolates of R. solani.     

Draft Genome Sequencing of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> Anastomosis Group 3 (AG3- PT) Causing Stem Canker and Black Scurf of Potato

Rhizoctonia solani is a soil-borne basidiomycete fungus with a necrotrophic lifestyle being classified into fourteen reproductively incompatible anastomosis groups (AGs). AG3-PT (a potato subgroup) is associated with quantitative and qualitative yield losses through stem canker and black scurf in potato. Here we present the first draft sequence of the R. solani [AG3-PT] strain RS-20 with a G-C content of 48.3%. It consists of 11,431 total predicted protein coding regions including 181 tRNA and 31 rRNA coding genes. The initial pBLAST revealed more than 97% hits among AG groups where as only 1.7% of genes hit with other organisms. The R. solani genome is found to be dominated with tri mer repeats. The genome-wide evolutionary studies revealed the close association of AG3-PT with AG3. The draft sequence represents a helpful resource not only for understanding the core genes involved in pathogenecity but also evolution and adaptive behaviour within the R. solani species complex.     

Comprehensive Characteristics of MicroRNA Expression Profile Conferring to Rhizoctonia solani in Rice

MicroRNAs(miRNAs) are about 22 nucleotides regulatory non-coding RNAs that play versatile roles in reprogramming plant responses to biotic and abiotic stresses. However, it remains unknown whether miRNAs confer the resistance to necrotrophic fungus Rhizoctonia solani in rice. To investigate whether miRNAs regulate the resistance to R. solani, we constructed 12 small RNA libraries from susceptible and resistant rice cultivars treated with water/pathogen at 5 h post inoculation(hpi), 10 hpi and 20 hpi, respectively. By taking the advantage of next-generation sequencing, we totally collected 400–450 known mi RNAs and 450–620 novel miRNAs from the libraries. Expression analysis of mi RNAs demonstrated different patterns for known and novel miRNAs upon R. solani challenge. Thirty-four mi RNA families were identified to be expressed specifically in rice, and most of them were involved in plant disease resistance. A particular Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis result revealed that a great majority of target genes of regulated miRNAs belonged to the pathway of plant-pathogen interaction. Moreover, miR444 b.2, miR531 a, mir1861 i, novel_miR1956 and novel_miR135 conferred response to R. solani infection confirmed by Northern blot. Our global understanding of miRNA profiling revealed that the regulation of mi RNAs may be implicated in the control of rice immunity to R. solani. Analysis of the expression of miRNAs will offer the community with a direction to generate appropriate strategies for controlling rice sheath blight disease.     

水稻立枯丝核菌G蛋白β亚基基因的克隆、表达及序列分析

根据Thanatephorus cucumeris G蛋白β亚基序列（AY884129）设计引物,对水稻立枯丝核菌AG 1IA的 G蛋白β亚基基因进行了克隆。PCR结果得到1条约为1.9 kb的扩增片段,包含1个约1.7 kb的完整开放阅读框,编码366个氨基酸。同源性检索发现该序列与大量Ｇ蛋白β亚基基因明显同源,一致性介于57.34%~88.14%。根据其推导cDNA序列设计引物进行RT PCR分析,发现该基因在对数生长期表达量最高,提示水稻立枯丝核菌AG 1IA G蛋白β亚基基因可能具有时空表达特性。     

Identification of sources of resistance to damping-off and early root/hypocotyl damage from Rhizoctonia solani in common bean (Phaseolus vulgaris L.)

Rhizoctonia solani causes economically important root and hypocotyl diseases in common bean throughout the world. Root health is a vital factor in plant development and root diseases would negatively influence water and nutrient uptake as well as cause direct stand reduction and root rot damage to the crop. An efficient common bean screening method to evaluate damping-off and early root/hypocotyl damage from R. solani was developed and used to identify dry bean lines with levels of resistance to this disease. Two sets of 163 and 111 lines previously evaluated for drought tolerance in Nebraska and Puerto Rico were evaluated for damping-off resistance and early root/hypocotyl damage under greenhouse conditions. Disease severity on plants was identified based on above-ground symptoms, seedling survival and root lesions using a rating scale of 1 (resistant) to 9 (susceptible). In the first set of lines representing commonly grown dry bean cultivars, germplasm and sources of damping-off resistance, the Rhizoctonia mean rating ranged from 1.7 to 3.9; Phaseolus vulgaris lines PI 310668 and PI 533249 had the highest damping-off resistance. In the second set of the best lines from a drought tolerance shuttle breeding program the Rhizoctonia mean rating was between 2.6 and 5.7. The availability of drought tolerant dry bean lines allowed the testing of the hypothesis that there was a correlation between selecting for drought tolerance and R. solani damping-off resistance. No correlation between mean disease rating and drought tolerance was found, but adapted dry bean lines such as NE14-08-176 released as SB-DT1, and NE14-08-225 were identified with moderate damping-off resistance and drought tolerance. Lines with both traits and other attributes will facilitate development of resistant bean cultivars to manage damping-off caused by R. solani.     

Antifungal activity in vitro of Aloe vera pulp and liquid fraction against plant pathogenic fungi

The leaf pulp of Aloe vera, designated as the gel, and the bitter, yellow liquid fraction have been tested against pathogens (bacteria and fungi) affecting human and plants. However, their activity for fungal control in commercial industrial crops has not been determined. The objectives of this study were to evaluate the inhibitory effect of Aloe pulp and liquid fraction on the mycelial growth of three phytopathogenic fungi and to determine the extract concentrations that can inhibit mycelial development. A. vera leaves were cut from plants grown under greenhouse conditions at the University Antonio Narro, disinfected with sodium hypochlorite, and separated in two groups. In the first group, the pulp was manually scraped out; in the second, a laboratory roll processor was used for the pulp and liquid fraction separation. Both types of extracts were pasteurized. Antifungal activity of pulp and liquid fraction was evaluated on the mycellium development of Rhizoctonia solani, Fusarium oxysporum, and Colletotrichum coccodes that were isolated from a potato crop by the hyphae point and monosporic techniques. The concentrations of the plant extract ranged from 0 to 10⁵ μl l⁻¹. Fungal plugs 0.4 mm in diameter were placed in Petri dishes with a potato–dextrose–agar (PDA) culture media, and treated with various concentrations of pulp or liquid fraction. The cultures were incubated at 24±2 °C and the radial growth of mycelia measured daily for 7 days. The antifungal effect was measured under a totally random design with four replications. The results showed an inhibitory effect of the pulp of A. Vera on F. oxysporum at 10⁴ μl l⁻¹ and over a long period. For the two types of Aloe fractions the activities were similar. Besides the liquid fraction reduced the rate of colony growth at a concentration of 10⁵ μl 1⁻¹ in R. solani, F. oxysporum, and C. coccodes. This is the first report of any Aloe liquid fraction activity against plant pathogenic fungi.     

Suppression of rice sheath blight disease using a heat stable culture filtrate from Streptomyces philanthi RM-1-138

Streptomyces philanthi RM-1-138, isolated from the rhizosphere soil of chili pepper in southern Thailand, was investigated for its antagonistic activity against phytopathogenic fungi. In dual culture on glucose yeast-malt extract (GYM) agar plates, this strain suppressed the mycelial growth of all seven plant pathogenic fungi tested (Rhizoctonia solani PTRRC-9, Pyricularia grisea PTRRC-18, Colletotrichum gloeosporioides NBCRSR-3, Colletotrichum capcisi NBCRSR-15, Ganoderma boninense NBCRSR-26, Fusarium fujikuroi PTRRC-16 and Bipolaris oryzae PTRRC-36) with an 82.2–89.2% inhibition and that was most pronounced on R. solani PTRRC-9. Heat treatment of the culture filtrate from growing R. solani PTRRC-9 at 40 °C, 60 °C, 80 °C, and 100 °C for 30 min and 121 °C for 15 min had no negative effect on the inhibitory activity against R. solani PTRRC-9 tested on both solid and liquid culture. The effective dose (>80% inhibition) of culture filtrate in liquid culture was at 5.0% (v/v) while it was at 10% (v/v) on the solid medium. This effectiveness was similar to those of the four chemical fungicides tested. The effect of S. philanthi RM-1-138 against R. solani PTRRC-9 was investigated using SEM and TEM. The compounds produced by S. philanthi RM-1-138 induced alterations to the cell-wall structure of R. solani PTRRC-9, that resulted in the loss of cytoplasm materials by partial lysis. The greenhouse experiment revealed that using either the culture filtrate or the autoclaved culture filtrate from S. philanthi RM-1-138 effectively suppressed rice sheath blight disease by up to 65.6 and 60.8%, respectively.     

Diverse no-till irrigated crop rotations instead of burning and plowing continuous wheat

Field burning of residue is a traditional management tool for irrigated wheat (Triticum aestivum L.) production in the Inland Pacific Northwest of the United States (PNW) that can result in reduced air quality. A 6-year no-till field experiment to evaluate two complete cycles of a 3-year irrigated crop rotation of winter wheat–spring barley (Hordeum vulgare L.)–winter canola (Brassica napus L.) was sown (i) directly into standing residue of the previous crop, (ii) after mechanical removal of residue and, (iii) after burning of residue. The traditional practice of continuous annual winter wheat sown after burning residue and inverting the topsoil with a moldboard plow was included as a check treatment. Over-winter precipitation storage efficiency (PSE) was markedly improved when residue was not burned or burned and plowed after grain harvest. Grain yield of winter wheat trended higher in all no-till residue management treatments compared to the check treatment. Average grain yields of spring barley and canola were not significantly different among the no-till residue management treatments. Winter canola failed in 5 of 6 years due to a combination of a newly identified Rhizoctonia damping-off disease caused by Rhizoctonia solani AG-2-1 and cold temperatures that necessitated replanting to spring canola. Six-year average net returns over total costs were statistically equal over all four systems. All systems lost from $358 to $396 ha^?1. Soil organic carbon (SOC) increased linearly each year with no-till at the 0–5 cm depth and accumulated at a slower rate at the 5–10 cm depth. Take-all of wheat caused by Gaeumannomyces graminis var. tritici was most severe in continuous annual winter wheat. The incidence and severity of Rhizoctonia on roots of wheat and inoculum of R. solani AG-8, was highest in the no-till treatments, but there was no grain yield loss due to this disease in any treatment. Residue management method had no consistent effect on Rhizoctonia root rot on barley. The annual winter grass downy brome (Bromus tectorum L.) was problematic for winter wheat in the standing and mechanically removed residue treatments, but was controlled in the no-till residue burned and the burn and plow check. Another winter annual grass weed, rattail fescue (Vulpia myuros L.), infested all no-till treatments. This was the first comprehensive and multidisciplinary no-till irrigated crop rotation study conducted in the Pacific Northwest.     

A Draft Genome Sequence Reveals the Helminthosporium solani Arsenal for Cell Wall Degradation

Helminthosporium solani is a slow-growing fungal pathogen belonging to the family Massarinaceae. It causes blemishes on potato tubers, affecting processing and fresh market trade. Despite its worldwide distribution, little is known about the biology of H. solani. Here we report the generation of a draft genome sequence of H. solani with an estimated genome size of ~35 megabases, for use in phylogenetic and pathogenicity studies of this fungal pathogen. This sequence is also the first reference genome within the family Massarinaceae. We identified a large suite of genes in the H. solani genome that encode putative cell wall degrading enzymes. Based on comparison with other genomes, we speculate that H. solani is a hemibiotroph or necrotroph. The presence of a large number of genes in the glycoside hydrolase (GH) 10 and 43 families, which aid in the hydrolysis of glucoronoarabinoxylan, also suggests that H. solani may be able to survive on grass hosts and indicates the need to re-examine the life cycle and host range of this pathogen.     

Suppression of PLRV Titer in transgenic Russet Burbank and Ranger Russet

Potato varieties Russet Burbank and Ranger Russet were transformed with a cDNA version of the 23 kD coat protein cistron (CP) of the potato leafroll virus (PLRV) using anAgrobacterium-mediated procedure. Clones were assayed for presence of thenpt- II and CP genes by Southern analysis, for expression of CP mRNA by Northern analysis, and for presence of PLRV coat protein in uninoculated and aphid inoculated plants by ELISA and Western blot analysis in uninoculated plants. Two putative Russet Burbank transformants were escapes, lacking eithernptll or CP, while one putative Ranger Russet transformant possessed thenpt- II gene but not the CP gene. In Russet Burbank and Ranger Russet, some transformants had statistically lower virus titer. The lowered titer was consistent throughout assays at three times during primary infection and one assay of secondary infection. The ranking of virus titer across all tests was statistically consistent. Transformants with the lowest secondary titers had virus contents 15 and 31 % of the titers of untransformed controls for Russet Burbank and Ranger Russet, respectively. The virus titer of the two Russet Burbank escapes and the Ranger Russet with only thenpt- II gene did not differ significantly from their respective untransformed controls.     

Control of Rhizoctonia solani fruit rot of tomatoes by Trichoderma harzianum Rifai

Application of Trichoderma harzianum, to soil or by coating tomato fruits, reduced Rhizoctonia solani fruit rot by up to 43% and 85%, respectively, under laboratory conditions. When mixed with naturally infested soil, Trichoderma reduced R. solani inoculum potential by 86% in field trials. It also significantly reduced fruit rot by 27–51%.